Methods of Determining Patient Response by Measurement of HER-3

ABSTRACT

The invention provides methods of measuring and/or quantifying the presence and/or amount of Her-3 and/or Her-3 in a complex in a sample. The invention also provides antibodies specific for Her-3.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 12/688,798, filed Jan. 15, 2010, which claims the benefit ofand priority under 35 USC §119(e) to U.S. Provisional Application No.61/176,630, filed May 8, 2009, U.S. Provisional Application No.61/187,962, filed Jun. 17, 2009, and U.S. Provisional Application No.61/145,029, filed Jan. 15, 2009. These applications are eachincorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

A biomarker generally has a characteristic that can be objectivelymeasured and evaluated as an indicator of normal biological processes,pathogenic processes or pharmacological responses to a therapeuticintervention. See Atkinson et al., 2001, Clin. Pharmacol. Ther.69:89-95. Biomarkers vary widely in nature, ease of measurement andcorrelation with physiological states of interest. See, e.g., Frank etal., 2003, Nature Reviews Drug Discovery 2:566-580. It is widelybelieved that the development of new validated biomarkers will lead bothto significant reductions in healthcare and drug development costs andto significant improvements in treatment for a wide variety of diseasesand conditions. Thus, a great deal of effort has been directed to usingnew technologies to find new classes of biomarkers. See, e.g., Petricoinet al., 2002, Nature Reviews Drug Discovery, 1:683-695; and Sidransky,2002, Nature Reviews Cancer 2:210-219; Ludwig and Weinstein, 2005,Nature Reviews Cancer 5:845-856; Lee et al., 2007, Adv. Cancer. Res.,96:269-298; Dhani and Siu, 2008, Cancer Metastasis Rev. 27:339-349;Carden et al., 2009, Clin. Pharmacol. Ther. 85:131-133.

The interactions of cell surface membrane components play crucial rolesin transmitting extracellular signals to a cell in normal physiology andin disease conditions. In particular, many types of cell surfacereceptors undergo dimerization, oligomerization or clustering inconnection with the transduction of an extracellular event or signalinto a cellular response, such as, e.g., proliferation, increased ordecreased gene expression or the like. See, e.g., George et al., 2002,Nature Reviews Drug Discovery 1:808-820; Mellado et al, 2001, Ann. Rev.Immunol. 19:397-421; Schlessinger, 2000, Cell 103:211-225; and Yarden,2001, Eur. J. Cancer 37:S3-S8. The role of such events in diseases, suchas cancer, has been the object of intense research and has led to thedevelopment of several new drugs and drug candidates. See, e.g., Herbstand Shin, 2002, Cancer 94:1593-1611; Yarden and Sliwkowski, 2001, NatureReviews Molecular Cell Biology 2:127-137; McCormick, 1999, Trends inCell Biology 9:53-56 (1999); and Blume-Jensen and Hunter, 2001, Nature411:355-365.

Expression levels of individual cell surface receptors, such as Her-2 inbreast cancer, have been used as biomarkers, especially to determinepatient prognosis or whether a patient will or will not respond tocertain treatments. In addition, oncogenic tyrosine kinases such asmembers of the epidermal growth factor receptor family have providedtargets for drug development. However, the tyrosine kinase inhibitorstargeted to EGFR and Her-2 have shown less clinical efficacy thananticipated from promising preclinical studies, which has led tointerest in other EGFR-family members, such as Her-3, in part forprognostic value as biomarkers and in part because of interactions withother family members, leading to potential new drug targets. SeeMenendez and Lupu, 2007, Breast Cancer Research 9:111; Lee-Hoeflich etal., 2008, Cancer Res 68:5878-5887; Fuchs et al., 2006, Anticancer Res.26:4397-4402; Sergina et al., 2007, Nature 445:437-441; and Tovey etal., 2006, J. Pathol. 210:358-362.

Her-3 is sometimes over-expressed in breast cancer, colorectal cancer,ovarian cancer, bladder cancer, prostate cancer, non-small cell lungcancer, melanoma, pharyngeal cancer, pancreatic cancer, esophagealcancer, glioma, biliary tract carcinoma, cholangiocarcinoma, gastriccancer, endometrial cancer, gall bladder cancer, squamous cell carcinomaor basal cell carcinoma. Conventional immunohistochemical (IHC) orfluorescence in situ hybridization (FISH) analyses have been used todetect Her-3 over-expression. Unfortunately, IHC and FISH have certainlimitations as diagnostic tools in that they are not necessarilyaccurate and also prone to different interpretations by differentlaboratory personnel. There are currently no methods for accuratelyassessing the level of Her-3. The advent of a quantitative method formeasuring Her-3 would facilitate the ability to accurately determine acancer patient's prognosis and/or whether a patient is likely to respondto a certain treatment. See Mosesson et al., 2004, Semin. Cancer. Biol.14:262-270.

SUMMARY OF THE INVENTION

In a first aspect, the invention is drawn to a method of measuringand/or quantifying the presence and/or amount of Her-3 and/or Her-3 in acomplex in a sample, the method comprising providing a sample anddetermining the presence and/or quantity of Her-3 and/or Her-3 in acomplex in the sample. In certain embodiments, the amount of Her-3 isabove a first threshold, such that the sample is stratified as having a“high” amount of Her-3 (e.g., either total Her-3 and/or Her-3 homodimersand/or Her-3 heterodimers). In some embodiments the first thresholdvalue for Her-3 is a total Her-3 (H3T) of ≧0.158 and a low Her-3 valueis below this threshold. Or, other ranges may be used depending upon thepatient cohort and/or the significant event being monitored. Thus, eachof the threshold values and/or threshold ranges described herein mayvary by about 0.5 log units or less on a log scale and/or 25% or less ona linear scale (i.e., be ≦25% larger and/or ≦25% smaller than thespecific ranges disclosed herein), or by about 20% or less, or by about15% or less, or by about 10% or less, or by about 5% or less.

In a preferred embodiment, the sample is a biological sample. In apreferred embodiment, the sample is a tissue sample. In a preferredembodiment, the sample is a fresh tissue sample, a fixed sample, afrozen sample or a lysate. In a preferred embodiment, the sample is atumor sample. In a preferred embodiment, the sample is a frozen tumortissue sample. In a preferred embodiment, the sample comprises a tumorlysate from a fresh or frozen tumor sample. In a preferred embodiment,the sample is an FFPE or solubilized FFPE sample. In a preferredembodiment, the sample comprises a breast cancer sample. In a certainembodiments, the breast cancer is early stage (i.e., adjuvant) breastcancer or metastatic breast cancer.

In certain embodiments of each of the methods and/or aspects of theinvention as disclosed herein, the method comprises detection of otherbiomarkers in the sample. For example, other biomarkers such as Her-2and/or p95 may be measured. Or, other biomarkers such as can be at leastone of FOXM1, PRAME, Bc12, STK15, CEGP1, Ki-67, GSTM1, CA9, PR, BBC3,NME1, SURV, GATA3, TFRC, YB-1, DPYD, GSTM3, RPS6 KB1, Src, Chk1, ID1,EstR1, p27, CCNB1, XIAP, Chk2, CDC25B, IGF1R, AK055699, P13KC2A, TGFB3,BAGI1, CYP3A4, EpCAM, VEGFC, pS2, hENT1, WISP1, HNF3A, NFKBp65, BRCA2,EGFR, TK1, VDR, Contig51037, pENT1, EPHX1, IF1A, CDH1, HIF1α, IGFBP3;CTSB, Her3 or DIABLO. In certain embodiments, the other biomarker can beVEGF, CD31, KDR, p95, or Her-2.

In certain embodiments of each of the methods and/or aspects of theinvention as disclosed herein, the level of Her-2 expression in thebreast cancer is high. In certain embodiments, high Her-2 expression isa log 10H2T≧ about 1.14-1.25. In certain embodiments, the high Her-2expression comprises expression that is very high and/or moderatelyhigh. In certain embodiments, the very high Her-2 expression is a log10H2T≧ about 1.84-2.21. In certain embodiments of each of the methodsdisclosed herein, the moderately high expression is between 1.14-1.25and 1.84-2.21 (i.e., ≧1.14-1.25 and ≦1.84-2.21). Or, other ranges may beused depending upon the patient cohort and/or the significant eventbeing monitored. Thus, each of the threshold values and/or thresholdranges described herein may vary by about 0.5 log units or less on a logscale and/or 25% or less on a linear scale (i.e., be ≦25% larger and/or≦25% smaller than the specific ranges disclosed herein), or by about 20%or less, or by about 15% or less, or by about 10% or less, or by about5% or less.

Also, in certain embodiments of each of the methods and/or aspects ofthe invention as disclosed herein, the level of p95 may be evaluated aseither high or low. In some embodiments the first threshold value forp95 is a total p95 value of ≧90 (on a linear scale) and a low p95 valueis below this threshold. Or, other ranges may be used depending upon thepatient cohort and/or the significant event being monitored. Thus, eachof the threshold values and/or threshold ranges described herein mayvary by about 0.5 log units or less on a log scale and/or 25% or less ona linear scale (i.e., be ≦25% larger and/or ≦25% smaller than thespecific ranges disclosed herein), or by about 20% or less, or by about15% or less, or by about 10% or less, or by about 5% or less.

In certain embodiments, if the level of Her-3 is high, the patient isless likely or unlikely to respond to the targeted therapy. In certainembodiments, if the level of Her-3 is low, the patient is more likely torespond to the targeted therapy. In certain embodiments, the therapy isa Her-acting agent. In certain embodiments, the therapy is at least oneof a Her-2 acting agent or a Her-3-targeted agent.

Thus, in certain embodiments of each of the methods and aspects of theinvention as disclosed herein, the method comprises measuring in abiological sample from the subject's cancer an amount of Her-2 and/orHer-2 homodimers, wherein if the amount of Her-2 and/or Her-2 homodimersis moderately high and Her-3 expression is low, then the patient islikely to respond to the Her-2 acting agent and/or the patient has along time course. In certain embodiments, the method comprises measuringin a biological sample from the subject's cancer an amount of Her-2and/or Her-2 homodimers, wherein if the amount of Her-2 and/or Her-2homodimers is moderately high and Her-3 expression is high, then thepatient is unlikely to respond to the Her-2 acting agent and/or thepatient has a short time course.

Additionally and/or alternatively, in certain embodiments of each of themethods and aspects of the invention as disclosed herein, the methodcomprises measuring in a biological sample from the subject's cancer anamount p95, wherein if the amount of p95 and Her-3 expression is low,then the patient is likely to respond to the therapeutically actingagent and/or the patient has a long time course. In an embodiment, thepatient also has a high (or moderately high) level of Her-2. In certainembodiments, the method comprises measuring in a biological sample fromthe subject's cancer an amount of Her-2 and/or Her-2 homodimers, whereinif the amount of Her-2 and/or Her-2 homodimers is high and/or moderatelyhigh and Her-3 expression and/or p95 expression is high, then thepatient is unlikely to respond to the Her-2 acting agent and/or thepatient has a short time course.

In a preferred embodiment, the sample is a blood, plasma or lymphsample. In a preferred embodiment, the blood or plasma sample containscirculating tumor cells. In a preferred embodiment, the sample comprisescell lines. In a preferred embodiment, the measurement may bequantitative across a wide dynamic range.

In a preferred embodiment, the method comprises mixing the sample with abinding compound and determining the presence and/or quantity of thebinding compound bound to Her-3 and/or Her-3 in a complex. In apreferred embodiment, the binding compound binds specifically to Her-3.In a preferred embodiment, the binding compound comprises an antibody.In a preferred embodiment, the antibody is raised against one of thepeptides having SEQ ID NOs:1-8, as set forth in Example 2 and shown inFIG. 2A. In a preferred embodiment, the antibody is a monoclonalantibody comprising (a) a light chain variable region comprising CDR1,CDR2 and CDR3 having sequences as set forth in SEQ ID NOs:13, 14 and 15,respectively, and (b) a heavy chain variable region comprising CDR1,CDR2 and CDR3 having sequences as set forth in SEQ ID NOs:16, 17 and 18,respectively; and/or a monoclonal antibody comprising (a) a light chainvariable region comprising CDR1, CDR2 and CDR3 having sequences as setforth in SEQ ID NOs:19, 20 and 21, respectively, and (b) a heavy chainvariable region comprising CDR1, CDR2 and CDR3 having sequences as setforth in SEQ ID NOs:22, 23 and 24, respectively. In a preferredembodiment, the antibody is the antibody with the amino acid sequencehaving SEQ ID NOs:9 and 11 as set forth in Table 1 (see DetailedDescription) for the light and heavy chains, respectively, and/or SEQ IDNOs:10 and 12 as set forth in Table 1 (see Detailed Description) for thelight and heavy chains, respectively. In a preferred embodiment, themethod comprises mixing (i) a sample that may contain Her-3 and/or Her-3in a complex; (ii) a proximity probe that is capable of binding Her-3,the proximity probe having an effective proximity and (iii) at least onebinding compound, the at least one binding compound being capable ofbinding Her-3 and having one or more signaling molecules attached,wherein binding of the proximity probe and the binding compound withinthe effective proximity produces a signal from the molecular tags thatcorrelates with the presence and/or quantity of Her-3 and/or Her-3 in acomplex. In a preferred embodiment, the proximity probe and/or bindingcompound is capable of binding specifically to Her-3 or the at least oneother analyte. In a preferred embodiment, the proximity probe and/orbinding compound further comprises an antibody and each antibody canbind to a specific epitope on Her-3. In a preferred embodiment, theantibody is raised against one of the peptides having SEQ ID NOs:1-8, asset forth in Example 2 and shown in FIG. 2A. In a preferred embodiment,the antibody is a monoclonal antibody comprising (a) a light chainvariable region comprising CDR1, CDR2 and CDR3 having sequences as setforth in SEQ ID NOs:13, 14 and 15, respectively, and (b) a heavy chainvariable region comprising CDR1, CDR2 and CDR3 having sequences as setforth in SEQ ID NOs:16, 17 and 18, respectively; and/or a monoclonalantibody comprising (a) a light chain variable region comprising CDR1,CDR2 and CDR3 having sequences as set forth in SEQ ID NOs:19, 20 and 21,respectively, and (b) a heavy chain variable region comprising CDR1,CDR2 and CDR3 having sequences as set forth in SEQ ID NOs:22, 23 and 24,respectively. In a preferred embodiment, the antibody is the antibodywith the amino acid sequence having SEQ ID NOs:9 and 11 as set forth inTable 1 (see Detailed Description) for the light and heavy chains,respectively, and/or SEQ ID NOs:10 and 12 as set forth in Table 1 (seeDetailed Description) for the light and heavy chains, respectively. In apreferred embodiment, the sample is a biological sample. In a preferredembodiment, the sample is a tissue sample. In a preferred embodiment,the sample is a fixed sample, a frozen sample or a lysate. In apreferred embodiment, the sample is a tumor sample. In a preferredembodiment, the sample is a frozen tumor tissue sample. In a preferredembodiment, the sample comprises a tumor lysate. In a preferredembodiment, the sample comprises a breast cancer sample. In a preferredembodiment, the sample is an FFPE sample or solubilized FFPE sample. Ina preferred embodiment, the sample is a blood, plasma or lymph sample.In a preferred embodiment, the blood or plasma sample containscirculating tumor cells. In a preferred embodiment, the sample comprisescell lines. In a preferred embodiment, the measurement may bequantitative across a wide dynamic range. In a preferred embodiment, thewide dynamic range is approximately 2 logs. In a more preferredembodiment, the wide dynamic range is about 1-1.5 logs in breast cancersamples. In a preferred embodiment, the method provides a quantitativecontinuum of Her-3 expression. In a preferred embodiment, themeasurement or quantity is sensitive to at least about 1000 receptorsper cell to about 200,000 receptors per cell as determined by accuracystudies utilizing well-characterized cell line models andcross-validating technologies such as ELISA and flow cytometry. In apreferred embodiment, the measurement or quantity is sensitive to atleast about 5000 receptors per cell to about 200,000 receptors per cell.In a preferred embodiment, the measurement or quantity is sensitive toat least about 10,000 receptors per cell to about 200,000 receptors percell. In a preferred embodiment, the measurement or quantity issensitive to at least about 25,000 receptors per cell to about 200,000receptors per cell. In a preferred embodiment, the measurement isspecific as determined using isotype control antibodies and comparisonwith conventional IHC methods.

In a further preferred embodiment, the proximity probe comprises anantibody and a first nucleic acid and the binding compound comprises anantibody and a second nucleic acid, wherein the first and the secondnucleic acids are complementary to each other and able to hybridize todetermine the effective proximity and produce the signal, directly orindirectly, through hybridization. In a preferred embodiment, theproximity probe and/or binding compound is capable of bindingspecifically to Her-3. In a preferred embodiment, the binding compoundand/or the proximity probe further comprises an antibody and eachantibody binds to a different epitope on Her-3. In a preferredembodiment, the antibody is raised against one of the peptides havingSEQ ID NOs:1-8, as set forth in Example 2 and shown in FIG. 2A. In apreferred embodiment, the antibody is a monoclonal antibody comprising(a) a light chain variable region comprising CDR1, CDR2 and CDR3 havingsequences as set forth in SEQ ID NOs:13, 14 and 15, respectively, and(b) a heavy chain variable region comprising CDR1, CDR2 and CDR3 havingsequences as set forth in SEQ ID NOs:16, 17 and 18, respectively; and/ora monoclonal antibody comprising (a) a light chain variable regioncomprising CDR1, CDR2 and CDR3 having sequences as set forth in SEQ IDNOs:19, 20 and 21, respectively, and (b) a heavy chain variable regioncomprising CDR1, CDR2 and CDR3 having sequences as set forth in SEQ IDNOs:22, 23 and 24, respectively. In a preferred embodiment, the antibodyis the antibody with the amino acid sequence having SEQ ID NOs:9 and 11as set forth in Table 1 (see Detailed Description) for the light andheavy chains, respectively, and/or SEQ ID NOs:10 and 12 as set forth inTable 1 (see Detailed Description) for the light and heavy chains,respectively. In a preferred embodiment, the sample is a biologicalsample. In a preferred embodiment, the sample is a tissue sample. In apreferred embodiment, the sample is a fixed sample, a frozen sample or alysate. In a preferred embodiment, the sample is a tumor sample. In apreferred embodiment, the sample is a frozen tumor tissue sample. In apreferred embodiment, the sample comprises a tumor lysate. In apreferred embodiment, the sample comprises a breast cancer sample. In apreferred embodiment, the sample is an FFPE sample or solubilized FFPEsample. In a preferred embodiment, the sample is a blood, plasma orlymph sample. In a preferred embodiment, the blood or plasma samplecontains circulating tumor cells. In a preferred embodiment, the samplecomprises cell lines. In a preferred embodiment, the measurement may bequantitative across a wide dynamic range. In a preferred embodiment, thewide dynamic range is about 2 logs. In a more preferred embodiment, thewide dynamic range is about 1-1.5 logs in breast cancer samples. In apreferred embodiment, the method provides a quantitative continuum ofHer-3 expression. In a preferred embodiment, the measurement or quantityis sensitive to at least about 1000 receptors per cell to about 200,000receptors per cell as determined by accuracy studies utilizingwell-characterized cell line models and cross-validating technologiessuch as ELISA and flow cytometry. In a preferred embodiment, themeasurement or quantity is sensitive to at least about 5000 receptorsper cell to about 200,000 receptors per cell. In a preferred embodiment,the measurement or quantity is sensitive to at least about 10,000receptors per cell to about 200,000 receptors per cell. In a preferredembodiment, the measurement or quantity is sensitive to at least about25,000 receptors per cell to about 200,000 receptors per cell. In apreferred embodiment, the measurement is specific as determined by usingisotype control antibodies and comparison with conventional IHC methods.

In a preferred embodiment, the proximity probe comprises a cleavingprobe that has a cleavage-inducing moiety and the at least one bindingcompound has one or more molecular tags attached to the binding compoundby a cleavable linkage, wherein the cleavable linkage may be cleavedwithin the effective proximity, producing a signal that correlates withthe presence and/or quantity of Her-3. In a preferred embodiment, thecleaving probe and/or binding compound is capable of bindingspecifically to Her-3. In a preferred embodiment, the binding compoundand/or the proximity probe further comprises an antibody and eachantibody binds to a different epitope on Her-3. In a preferredembodiment, the antibody is raised against one of the peptides havingSEQ ID NOs:1-8, as set forth in Example 2 and shown in FIG. 2A. In apreferred embodiment, the antibody is a monoclonal antibody comprising(a) a light chain variable region comprising CDR1, CDR2 and CDR3 havingsequences as set forth in SEQ ID NOs:13, 14 and 15, respectively, and(b) a heavy chain variable region comprising CDR1, CDR2 and CDR3 havingsequences as set forth in SEQ ID NOs:16, 17 and 18, respectively; and/ora monoclonal antibody comprising (a) a light chain variable regioncomprising CDR1, CDR2 and CDR3 having sequences as set forth in SEQ IDNOs:19, 20 and 21, respectively, and (b) a heavy chain variable regioncomprising CDR1, CDR2 and CDR3 having sequences as set forth in SEQ IDNOs:22, 23 and 24, respectively. In a preferred embodiment, the antibodyis the antibody with the amino acid sequence having SEQ ID NOs:9 and 11as set forth in Table 1 (see Detailed Description) for the light andheavy chains, respectively, and/or SEQ ID NOs:10 and 12 as set forth inTable 1 (see Detailed Description) for the light and heavy chains,respectively. In a preferred embodiment, the sample is a biologicalsample. In a preferred embodiment, the sample is a tissue sample. In apreferred embodiment, the sample is a fixed sample, a frozen sample or alysate. In a preferred embodiment, the sample is a tumor sample. In apreferred embodiment, the sample is a frozen tumor tissue sample. In apreferred embodiment, the sample comprises a tumor lysate. In apreferred embodiment, the sample comprises a breast cancer sample. In apreferred embodiment, the sample is an FFPE sample or solubilized FFPEsample. In a preferred embodiment, the sample is a blood, plasma orlymph sample. In a preferred embodiment, the blood or plasma samplecontains circulating tumor cells. In a preferred embodiment, the samplecomprises cell lines. In a preferred embodiment, the measurement may bequantitative across a wide dynamic range. In a preferred embodiment, thewide dynamic range is about 2 logs. In a more preferred embodiment, thewide dynamic range is about 1-1.5 logs in breast cancer samples. In apreferred embodiment, the method provides a quantitative continuum ofHer-3 expression. In a preferred embodiment, the measurement or quantityis sensitive to at least about 1000 receptors per cell to about 200,000receptors per cell as determined by accuracy studies utilizingwell-characterized cell line models and cross-validating technologiessuch as ELISA and flow cytometry. In a preferred embodiment, themeasurement or quantity is sensitive to at least about 5000 receptorsper cell to about 200,000 receptors per cell. In a preferred embodiment,the measurement or quantity is sensitive to at least about 10,000receptors per cell to about 200,000 receptors per cell. In a preferredembodiment, the measurement or quantity is sensitive to at least about25,000 receptors per cell to about 200,000 receptors per cell. In apreferred embodiment, the measurement is specific as determined by usingisotype control antibodies and comparison with conventional IHC methods.

In a second aspect, the invention is drawn to a method for determiningwhether a subject with a cancer is likely to respond to treatment with atargeted therapy, for predicting a time course of disease and/or forpredicting the probability of a significant event in the time course ofthe subject's cancer, comprising measuring in a biological sample fromthe subject's cancer an amount of Her-3, wherein the method is dependenton the level of Her-3. In certain embodiments, if the level of Her-3 ishigh, the patient is less likely or unlikely to respond to the targetedtherapy. In certain embodiments, if the level of Her-3 is low, thepatient is more likely to respond to the targeted therapy. In certainembodiments, as described in more detail herein, the therapy is a Heracting agent. In further embodiments, the therapy is at least one of aHer-2 acting agent or a Her-3-targeted agent. In certain embodiments,the amount of Her-3 is above a first threshold, such that the sample isstratified as having a “high” amount of Her-3 (e.g., either total Her-3and/or Her-3 homodimers and/or Her-3 heterodimers). In some embodimentsthe first threshold value for her-3 is a total Her-3 (H3T) of ≧0.158 anda low Her-3 value is below this threshold. Or, other ranges may be useddepending upon the patient cohort and/or the significant event beingmonitored. Thus, each of the threshold values and/or threshold rangesdescribed herein may vary by about 0.5 log units or less on a log scaleand/or 25% or less on a linear scale (i.e., be ≦25% larger and/or ≦25%smaller than the specific ranges disclosed herein), or by about 20% orless, or by about 15% or less, or by about 10% or less, or by about 5%or less.

In a preferred embodiment, the subject's cancer is breast cancer,colorectal cancer, ovarian cancer, bladder cancer, prostate cancer,non-small cell lung cancer, melanoma, pharyngeal cancer, pancreaticcancer, esophageal cancer, glioma, biliary tract carcinoma,cholangiocarcinoma, gastric cancer, endometrial cancer, gall bladdercancer, squamous cell carcinoma or basal cell carcinoma. In a preferredembodiment, the subject's cancer is breast cancer, melanoma, synovialcarcinoma, colorectal cancer or ovarian cancer. In a preferredembodiment, the subject's cancer is a Her-2 positive breast cancer. In acertain embodiments, the breast cancer is early stage (i.e., adjuvant)breast cancer or metastatic breast cancer.

As noted above, in certain embodiments, the method comprises detectionof other biomarkers in the sample. For example, other biomarkers such asHer-2 and/or p95 may be measured. Or, other biomarkers such as can be atleast one of FOXM1, PRAME, Bc12, STK15, CEGP1, Ki-67, GSTM1, CA9, PR,BBC3, NME1, SURV, GATA3, TFRC, YB-1, DPYD, GSTM3, RPS6 KB1, Src, Chk1,ID1, EstR1, p2′7, CCNB1, XIAP, Chk2, CDC25B, IGF1R, AK055699, P13KC2A,TGFB3, BAGI1, CYP3A4, EpCAM, VEGFC, pS2, hENT1, WISP1, HNF3A, NFKBp65,BRCA2, EGFR, TK1, VDR, Contig51037, pENT1, EPHX1, IF1A, CDH1, HIF1α,IGFBP3; CTSB, Her3 or DIABLO. In certain embodiments, the otherbiomarker can be VEGF, CD31, KDR, p95, or Her-2.

In certain embodiments, the level of Her-2 expression in the breastcancer is high. In certain embodiments, high Her-2 expression is a log10H2T≧ about 1.14-1.25. In certain embodiments, the high Her-2expression comprises expression that is very high and/or moderatelyhigh. In certain embodiments, the very high Her-2 expression is a log10H2T≧ about 1.84-2.21. In certain embodiments of each of the methodsdisclosed herein, the moderately high expression is between 1.14-1.25and 1.84-2.21 (i.e., ≧1.14-1.25 and ≦1.84-2.21). Or, other ranges may beused depending upon the patient cohort and/or the significant eventbeing monitored. Thus, each of the threshold values and/or thresholdranges described herein may vary by about 0.5 log units or less on a logscale and/or 25% or less on a linear scale (i.e., be ≦25% larger and/or≦25% smaller than the specific ranges disclosed herein), or by about 20%or less, or by about 15% or less, or by about 10% or less, or by about5% or less.

Also, in certain embodiments, the level of p95 may be evaluated aseither high or low. In some embodiments the first threshold value forp95 is a total p95 value of > or ≧90 (on a linear scale) and a low p95value is below this threshold. Or, other ranges may be used dependingupon the patient cohort and/or the significant event being monitored.Thus, each of the threshold values and/or threshold ranges describedherein may vary by about 25% or less (i.e., be ≦25% larger and/or ≦25%smaller than the specific ranges disclosed herein), or by about 20% orless, or by about 15% or less, or by about 10% or less, or by about 5%or less.

In certain embodiments, if the level of Her-3 is high, the patient isless likely or unlikely to respond to the targeted therapy. In certainembodiments, if the level of Her-3 is low, the patient is more likely torespond to the targeted therapy. In certain embodiments, the therapy isa Her-acting agent. In certain embodiments, the therapy is at least oneof a Her-2 acting agent or a Her-3-targeted agent.

Thus, in certain embodiments, the method comprises measuring in abiological sample from the subject's cancer an amount of Her-2 and/orHer-2 homodimers, wherein if the amount of Her-2 and/or Her-2 homodimersis moderately high and Her-3 expression is low, then the patient islikely to respond to the Her-2 acting agent and/or the patient has along time course. In certain embodiments, the method comprises measuringin a biological sample from the subject's cancer an amount of Her-2and/or Her-2 homodimers, wherein if the amount of Her-2 and/or Her-2homodimers is moderately high and Her-3 expression is high, then thepatient is unlikely to respond to the Her-2 acting agent and/or thepatient has a short time course.

Additionally and/or alternatively, in certain embodiments, the methodcomprises measuring in a biological sample from the subject's cancer anamount p95, wherein if the amount of p95 and Her-3 expression is low,then the patient is likely to respond to the therapeutically actingagent and/or the patient has a long time course. In an embodiment, thepatient also has a high (or moderately high) level of Her-2. In certainembodiments, the method comprises measuring in a biological sample fromthe subject's cancer an amount of Her-2 and/or Her-2 homodimers, whereinif the amount of Her-2 and/or Her-2 homodimers is high and/or moderatelyhigh and Her-3 expression and/or p95 expression is high, then thepatient is unlikely to respond to the Her-2 acting agent and/or thepatient has a short time course.

In a preferred embodiment, the targeted therapy is at least one Herfamily-targeted agent. In a preferred embodiment, the Herfamily-targeted agent is a multi- or single-targeted agent. In apreferred embodiment, the multi-targeted agent is a dual kinaseinhibitor or a bispecific antibody. In a preferred embodiment, the Herfamily-targeted agent is trastuzumab, lapatinib or pertuzumab. In apreferred embodiment, the at least one Her family-targeted agent is atleast two agents, wherein the at least two agents are one or moreHer-2-targeted monoclonal antibodies and/or EGFR-targeted monoclonalantibodies and/or an EGFR and Her-2 dual kinase inhibitor. In apreferred embodiment, the monoclonal antibody is trastuzumab. In apreferred embodiment the EGFR-targeted monoclonal antibody is cetuximabor panitumumab. In a preferred embodiment, the dual kinase inhibitor islapatinib, erlotinib or gefitinib. In a preferred embodiment, thetargeted therapy is a Her-3 or Her-3 signaling pathway acting agent. Ina preferred embodiment, the Her-3 or Her-3 signaling pathway targetedagent is a Her-3 monoclonal antibody, a Her-3 dimerization inhibitor, aHer-3 phosphorylation inhibitor and/or an inhibitor of a Her-3 signalingpathway member selected from the group consisting of PI3K, Akt, mTOR andERK1/2. In a preferred embodiment, likeliness to respond, likeliness tohave a long time course and/or likeliness to have a significant event ismeasured as an overall survival rate, as time to progression, asdisease-free survival, as progression-free survival, and/or as objectivetumor response using the RECIST criteria.

In a preferred embodiment, whether the cancer is Her-2 positive isdetermined by IHC, FISH, CISH, quantitative mRNA, a hybridization array,or VERATAG®. In a preferred embodiment, determining the level of Her-3is performed using IHC, FISH, CISH, quantitative mRNA, hybridizationarray, or VERATAG®.

In a preferred embodiment, the method further comprises determining thata subject is afflicted with a Her-2 positive cancer that is unlikely torespond to treatment according to a method of the invention, thenadvising a medical professional of the treatment option of administeringto the subject an effective amount of a different therapeutic agent.

In a third aspect, the invention is drawn to a purified antibody thatbinds to Her-3. In a preferred embodiment, the antibody is a polyclonalantibody or a monoclonal antibody. In a preferred embodiment, theantibody is a monoclonal antibody. In a preferred embodiment, theantibody is raised against one of the peptides having SEQ ID NOs:1-8, asset forth in Example 2 and shown in FIG. 2A. In a preferred embodiment,the antibody is a monoclonal antibody comprising (a) a light chainvariable region comprising CDR1, CDR2 and CDR3 having sequences as setforth in SEQ ID NOs:13, 14 and 15, respectively, and (b) a heavy chainvariable region comprising CDR1, CDR2 and CDR3 having sequences as setforth in SEQ ID NOs:16, 17 and 18, respectively; and/or a monoclonalantibody comprising (a) a light chain variable region comprising CDR1,CDR2 and CDR3 having sequences as set forth in SEQ ID NOs:19, 20 and 21,respectively, and (b) a heavy chain variable region comprising CDR1,CDR2 and CDR3 having sequences as set forth in SEQ ID NOs:22, 23 and 24,respectively. In a preferred embodiment, the antibody is the antibodywith the amino acid sequence having SEQ ID NOs:9 and 11 as set forth inTable 1 (see Detailed Description) for the light and heavy chains,respectively, and/or SEQ ID NOs:10 and 12 as set forth in Table 1 (seeDetailed Description) for the light and heavy chains, respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the levels of Her-3 expression as determined by a Her-3ELISA kit (R&D Systems, Inc.) in several stably-transfected clones ofHEK 293 (human embryonic kidney cells) transfected with an HER3expression vector. The construction of the expression vector isdescribed in Example 1. One clone, 293H3-Clone 1, expressed high levelsof HER3 and was selected as a control for use in the optimizedHER3VERATAG® assay.

FIG. 2A lists peptide sequences used to immunize mice to raiseHer-3-specific antibodies, wherein SEQ ID NO:1 isLGSALSLPVLNRPRGTGQSLLSP; SEQ ID NO:2 is SAYHSQRHSLLTPVTPLSP; SEQ ID NO:3is VGSDLSASLGSTQSCPLHPVPI; SEQ ID NO:4 is CQGPGHQAPHVHYARLKTLRS; SEQ IDNO:5 is LEEVELEPELDLDLDLEAE; SEQ ID NO:6 is CFDNPDYWHSRLFPKANA; SEQ IDNO:7 is CPDYWHSRLFPKANAQRT; and SEQ ID NO:8 is CFPKANAQRT. The peptidesequences represent different epitopes from the C-terminal region ofHer-3 (the length of each peptide and the position relative to theN-terminus of the protein are shown). Antibodies raised against eachpeptide are listed in the fourth column. Each antibody has beenconfirmed to test positive in ELISA, IHC, and VERATAG® assays. FIG. 2Bshows the results of IHC studies in which two cell lines were screenedwith B9A11, an Her-3-specific antibody raised against one of the Her-3peptides, an Her-2-specific antibody Ab8 (HerceptTest), and a controlantibody ITC-IgG2a. One cell line, SKOV3 (upper panels), is known toexpress high levels of HER2 and low levels of HER3. The other cell line,293H3-clone1 (lower panels), is the stablytransfected cell linedescribed in Example 1 and shown in FIG. 1 to over-express HER3 butwhich expresses low levels of HER2. The IHC results show a strong signalof B9A11 with the 293H3-clone 1 cells but not the SKOV3 cells, asexpected.

FIG. 3 shows HER3 levels as determined by VERATAG® in FFPE blocks from 4different cell lines, cross-validated with data from three other assays(IHC, ELISA, and flow cytometry). Cell lines expressing varying levelsof HER3 were chosen for these studies: 293H3-Clone 1, MDA-MB-453,MDAMB-468 and SKOV3 (the latter 3 from ATCC). The cell lines were chosento represent HER3 receptor levels that spanned greater than 2 logs. FFPEblocks were prepared as described in Example 3 for testing with theHER3VERATAG® assay and IHC. A portion of the cells from the same lotwere tested for HER3 receptor number using flow cytometry. Further, awhole cell lysate was prepared from the same lot of cells forquantifying HER3 with an ELISA kit (Human ErbB3-DuoSet ELISA: R&DSystems, Inc.). The data shows a wide dynamic range for the VERATAG®assay, with results consistent with all three other methodologies.

FIG. 4 shows examples of patient tumor samples in which aBoard-certified pathologist has circled the tumor area.

FIG. 5 shows the equipment used and the work flow of the HER3VERATAG®assay. FFPE samples are first deparaffinized and rehydrated using aseries of solvents (top panel 1). Antigen retrieval is accomplishedusing 1×DAKO (Lab Vision) in a pressure cooker (top panel 2). Thesamples are then rinsed with water and a hydrophobic pen is used to drawa circle around the sample, retaining reagents on the slide. The samplesare then blocked and treated with a mixture of VERATAG®-conjugated Ab-6(Lab Vision) and biotin-conjugated B9A11 (top panel 3). After incubationand washing, streptavidin-conjugated methylene blue reagent is added,incubated and washed, and then illumination buffer containingfluorescein and two capillary electrophoresis internal markers (MF andML, marker first and last, respectively) is added. The bound VERATAG® isreleased using an LED array, which photoactivates cleavage of theVERATAG® (top panel 4). VERATAG® intermediates are reduced to aquantifiable form using sodium borohydride and the VERATAG® reportersare separated and detected using capillary electrophoresis (ABI3130 CEinstrument, top panel 5).

FIG. 6 shows the results from an experiment designed to identify theoptimal antibody concentration for maximizing the dynamic range of theVERATAG® assay. Cell lines spanning the entire dynamic range of theassay were chosen: 293H3-Clone 1, MDA-MB-453, MDA-MB-468, MDA-MB-231 andSKOV3. The concentration of the antibodies B9A11-biotin and Ab-6 Pro-11were varied (column 1 of the table) as follows: 1 mg/mL B9A11-biotin and1 mg/mL Ab-6 Pro-11, 1 mg/mL B9A11-biotin and 2 mg/mL Ab-6 Pro-11 and 2mg/mL B9A11-biotin and 1 mg/mL Ab-6 Pro-11, in rows 4, 5 and 6,respectively. The results for each cell line are shown in the bar graph.Expected fold changes for pair-wise comparisons were based on HER3 flowcytometry and ELISA results from the same cell line FFPE blockpreparation. An optimal concentration of 2 mg/mL B9A1 1-biotin and 1mg/mL Ab-6 Pro-11 was chosen (circled) for best performance based on theaccurate detection of HER3 as compared with the expected fold changesshown in row 2 of the table. The dynamic range shown here isapproximately 2 logs.

FIG. 7 shows the accuracy of the HER3VERATAG® assay using threesuccessful replicates from four well-characterized cell lines(293H3-Clone 1, MDA-MB-453, MDA-MB-468 and SKOV3). The VERATAG®measurements were compared with in-house generated flow cytometry andELISA data. 100% of the results matched the in-house data from flowcytometry and ELISA in that 293H3-clone 1>MDA-MB-453>MDA-MB-468>SKOV3.No overlap was observed between signal levels for any of the four cellline samples.

FIG. 8 demonstrates the sensitivity of the HER3VERATAG® assay. One batchcontaining 8 replicates of the low HER3 expression control cell line,MDA-MB-468, was compared with 8 replicates of the low/negative HER3expression control cell line, SKOV3, to determine sensitivity. All ofthe pairwise comparisons (64/64) between MDA-MB-468 and SKOV3 resultedin MDA-MB-468 having higher levels of HER3 than SKOV3.

FIG. 9 shows the inter-assay reproducibility of the HER3VERATAG® assay.Eight separate HER3 total VERATAG® assays were performed on the fourwell-characterized cell lines, 293H3-Clone 1, MDA-MB-453, MDA-MB-468 andSKOV3, using different CE illuminators, several operators and ondifferent days over a 4 week period. Following batch normalizationprocedures, the data was compared across the 8 batches to ascertainreproducibility. The coefficient of variability across the dynamic rangewas between 8 and 15%. Values are represented as the Log₁₀ normRPA,which is the log of the normalized relative peak area/tumor area andthen batch normalized using expected values.

FIG. 10 shows the precision of the HER3VERATAG® assay. The intra-assayreproducibility of the HER3VERATAG® assay was demonstrated by comparingthe performance of 15 replicates of each of the 3 control cell lines,293H3-Clone 1, MDA-MB-453 and MDA-MB468. Pairwise comparisons were madeof the 15 replicates in each batch to determine precision. 95% of the293H3-Clone 1 data was within 1.2-fold and 95% of the MDA-MB-468 data iswithin 1.37-fold. The VERATAG® data (shown in normalized RPA) for the 15replicates of each cell line is shown by the 15 bars on the right ofeach panel. The control data for the 3 cell lines expressing moderate tohigh levels of HER3 and a low/negative HER3 expressing cell line, SKOV3,are shown on the left of each panel.

FIG. 11 shows the linearity of the HER3VERATAG® assay using differentsample sizes. Samples of diminishing size (1×, ½×, ¼×, 1/16×) from eachof the 3 well-characterized cell lines, 293H3-Clone 1, MDA-MB-453 andMDA-MB-468, were tested in the VERATAG® assay and the data was comparedin a pairwise manner to assess the linearity of the assay. TheMDA-MB-453 cell line shows linearity down to approximately 1/16^(th) ofthe original sample size; MDA-MB-468 shows linearity down toapproximately ½ of the original sample size.

FIG. 12 shows the specificity of the HER3VERATAG® assay as determined byisotype controls. Isotype control antibodies were tested in the VERATAG®assay format to ascertain the non-specific background of the assay. Forthe HER3Ab-6-Pro11 antibody, the isotype control was IgG1-Pro11. For theHER3B9A11-biotin antibody, the isotype control was IgG1-biotin. Signalderived using these isotype controls is not antigen-specific andtherefore represents non-specific background. In the each panel, theVERATAG® results are shown for the normal assay format using theHER3Ab6-Pro11 and B9A11-biotin antibodies in the bars labeled “control.”In the left panel, the VERATAG® data is shown for the HER3Ab6-Prol 1 andIgG1-biotin antibodies in the bars labeled “IgG-bio.” In the rightpanel, the VERATAG® data is shown for the HER3B9A11-biotin andIgG1-Pro11 antibodies in the bars labeled “IgG-Pro11.” Each antibodypairing was tested on an array of FFPE samples including standard cellline controls (293H3-Clone 1, MDA-MB-453, MDA-MB468, SKOV3 and T47D) aswell as several tumor samples (41776B1, 32712A2, 30345C2, 106305A2, and106341A2). Units are normalized RPA*IB/TA.

FIG. 13 shows VERATAG® data on tumor samples from the InternationalSerum Her-2/neu Study Group trial. This cohort of patients (n=105) wasprospectively observed during trastuzumab treatment between 1999 and2006. All patients were determined to be Her-2 positive by either IHC orFISH and had not been exposed to trastuzumab prior to the study. Thesamples were evaluated for HER3 levels using the HER3VERATAG® assay ineight separate batches. The results are shown in the 8 panels in thisfigure. Each panel also includes results for 5 control cell lines:293H3-Clone 1, MDA-MB-453, MDA-MB-468, SKOV3 and T47D (shown on the leftof each panel). Results are shown in log₁₀ normalized RPA units.

FIG. 14 shows the data from positional scanning analyses used todetermine the optimal cut-off for trastuzumab (i.e., Herceptin®)response. In the left panel, patients above the statisticallysignificant cut-off (see arrow) had an unfavorable time to progression(TTP) compared to patients below the cut-off (hazard ratio=2.3;p=0.0004). In the right panel, a significant cutoff could not bedetermined, but using the cut-off found for TTP, a trend for worseoverall survival (OS) in patients above the cut-off (indicated by thearrow) was observed (hazard ratio=1.7; p=0.059).

FIG. 15 shows Kaplan-Meier plots for a cohort of 82 trastuzumab-treatedpatients stratified first by total HER2 levels (H2T) and then furtherstratified by total HER3 levels (H3T). In the upper left panel, aKaplan-Meier plot shows the percent of patients with progression-freesurvival (months) for two groups of patients subdivided (based on apreviously reported cut-off) into HER2-normal and HER-2 over-expressinggroups. The HER2 over-expressing group was then further subdivided,using the cut-off shown in FIG. 14, into two subgroups based on thelevel of HER3. The Kaplan-Meier plot of these two subgroups is shown inthe upper right panel. The lower panel shows three sets of results fromthe upper panels: the normal HER2 group (log(H2T)<1.14), the HER2-high,HER3-low group (log(H2T)>1.14, H3T<0.158) and the HER2-high, HER3-highgroup (log(H2T)>1.14, H3T>0.158). Univariate Cox proportional hazardsanalyses examining the HER3-over-expressing subgroup identified H3T(high vs low) as the most significant predictor of time to progression(TTP; HR=2.98, p=0.0004).

FIG. 16 shows the results of HER3VERATAG® assays in synovial carcinoma,colon cancer and ovarian cancer. The top panel shows the results for apanel of synovial carcinoma samples. The 4 bars on the left are controlsamples (293H3-Clone 13, MDA-MB-453, MDAMB-468 and SKOV3, from left toright, respectively). The middle panel shows the results for a panel ofcolon cancer samples. The same control cell lines are used (shown in theleft 4 bars). The lower panel shows results comparing HER2 expression toHER3 expression in a panel of ovarian tumor samples. Results are shownin normalized RPA units. The dynamic range of these tumor samples rangesfrom 0.5-1.5, depending on the cancer.

FIG. 17 shows that with the High HER2 (H2T) group (log 10H2T>1.25,or >13.8 on the linear scale), the ability to subgroup patients intodifferent groups based on High or low p95 (p95 >90 or <=90) and high orlow HER3 (H3T) allows further stratification of clinical outcomes asmeasured by median TTP. Univariate KM analysis with the p95 and H3Tsubgroups combined, gives the results in the KM plots in this Figure.These data suggest that HER2-positive breast cancer patients as assessedby HERmark®/VERATAG® (i.e., high H2T, ie log 10H2T>1.25, or >13.8 on thelinear scale) can be classified into at least 4 sub-groups withdifferent outcomes following trastuzumab treatment. In the KM, the groupwith low H3T (in generation 1 of the HER3 assay, H3T<0.158) and low p95(p95<90) has a median TTP of 15.0 months, compared with 9.3 months forthe group with low H3T (H3T<0.158) and high p95 (p95>90); compared with6.4 months for the high H3T group (H3T>0.158 in generation 1 of the HER3assay) and low p95 group, and compared with 3.2 months for the high H3T(H3T>0.158) and high p95 (p95>90). The trend for the differences amongthe groups is significant (p<0.0001).

FIG. 18 shows Kaplan-Meier (KM) analyses comparing the percentprogression free on the y axis over time on the x axis (time toprogression, TTP) of various subgroups from the Lipton cohort, asdefined by the combined VERATAG® measurements of HER2 total (H2T high orlow), p95HER2 (p95 high or low), and HER3Total (H3T) high (H3T>0.158, inGeneration 1 of the H3T assay) or H3T low (H3T<=0.158 in Generation 1 ofthe assay). Cut-offs were identified by lowest p-value in a positionalscanning analysis. H2T high=(log 10H2T>1.25 or on a linearscale, >13.8). Low H2T=log 10H2T<=1.25 or on a linear scale, <=13.8. p95low=p95<=90 and p95 high=p95>90 (on a linear scale), and H3T hi >0.158on a linear scale, and H3Tlo<=0.158 on a linear scale). KM analysesdemonstrated that patients who were FISH positive, H2T high, p95 lo(low) and H3T lo (low) had a median TTP of 14.7 months, compared withthe 4 other groups that did not fare as well. Three groups with nearlysuperimposable lines (i.e., FISHnegative/H2T10 group−median TTP=4.5,FISH positive/H2Tlo (low) group-median TTP=3.7, and FISHpositive/H2Thi(high)/p95hi (high)/H3T hi (high)−median TTP=3.2) all had shorter medianTTP than the group FISH positive/H2Thigh/p95lo/H3T10−median TTP=15). Thegroup defined as FISH-positigve/H2Thi (high)/and p95 or H3Thigh had amedian TTP=9.3, which was in between the group with the best median TTP(FISH positive/H2Thigh/p95lo/H3Tlo) and the 3 groups in red/blue andblack. Thus, HERmark assay identified multiple subgroups of HER2positive patients with varying clinical outcomes as measured by TTP ontrastuzumab-based therapy. Neither the magnitude of HER2 over-expressionnor the outcome for these subgroups was predictable by FISH/CEP17 copynumber. HER2FISH positive MBC patients with high p95 and/or high H3T mayrepresent subsets of patients with de novo resistance to trastuzumab.While the applicants do not wish to be confined to any particularmechanistic theory, possible mechanisms that may account for the poorresponse to trastuzumab observed in these subgroups may includeinsufficient trastuzumab and/or lack of trastuzumab binding target(i.e., p95) and increased signaling via formation of heterodimers thatare not completely suppressed by trastuzumab.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the terms “embodiment” and “aspect” are usedinterchangeably.

“Antibody” means an immunoglobulin that binds to, and is thereby definedas complementary with, a particular spatial and polar organization ofanother molecule. The antibody can be monoclonal, polyclonal orrecombinant and can be prepared by techniques that are well known in theart such as immunization of a host and collection of sera (polyclonal)or by preparing continuous hybrid cell lines and collecting the secretedprotein (monoclonal) or by cloning and expressing nucleotide sequencesor mutagenized versions thereof coding at least for the amino acidsequences required for binding. Antibodies may include a completeimmunoglobulin or fragment thereof, which immunoglobulins include thevarious classes and isotypes, such as IgA, IgD, IgE, IgG1, IgG2a, IgG2band IgG3, IgM, etc. Fragments thereof may include Fab, Fv and F(ab′)2,Fab′ and the like. Antibodies may also be single-chain antibodies,chimeric antibodies, humanized antibodies or any other antibodyderivative known to one of skill in the art that retains bindingactivity that is specific for a particular binding site. In addition,aggregates, polymers and conjugates of immunoglobulins or theirfragments can be used where appropriate so long as binding affinity fora particular binding site is maintained. Guidance in the production andselection of antibodies and antibody derivatives for use inimmunoassays, including such assays employing releasable molecular tags(as described below) can be found in readily available texts andmanuals, e.g., Harlow and Lane, 1988, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, New York; Howard and Bethell, 2001,Basic Methods in Antibody Production and Characterization, CRC Press;Wild, ed., 1994, The Immunoassay Handbook, Stockton Press, New York.

“Binding compound” shall refer to a molecule capable of binding toanother molecule of interest. A binding compound may be an antibody, apeptide, a peptide or non-peptide ligand for a cell surface receptor, aprotein, an oligonucleotide, an oligonucleotide analog, such as apeptide nucleic acid, a lectin or any other molecular entity that iscapable of specifically binding to a target molecule or complex. In oneembodiment, the target molecule is a protein or protein complex. Inanother embodiment, a binding compound further comprises a proximityprobe. In one embodiment, a binding compound comprises one or moremolecular tags attached to a binding moiety. In another embodiment, asecond binding compound may be bound to the binding compound andmeasured or quantified as a correlative for the presence of the bindingcompound, which is bound to the target protein. As another specificexample, either the first or second binding compound may generate aneffector molecule that acts in conjunction with a proximity probe withan effective proximity, producing a signal that correlates with thepresence of the target protein. Further, in another embodiment, bindingcompounds may have molecular tags that interact with one another withinan effective proximity to form a complex that generates a signal or canbe detected and measured in a manner that correlates with the presenceof the target protein. More specifically, the target protein or complexmay be Her-3 or Her-3 in a complex.

“Binding moiety” means any molecule to which molecular tags can bedirectly or indirectly attached that is capable of binding to ananalyte. Binding moieties include, but are not limited to, antibodies,peptides, proteins, nucleic acids and organic molecules having amolecular weight of up to about 1000 daltons and containing atomsselected from the group consisting of hydrogen, carbon, oxygen,nitrogen, sulfur and phosphorus. Preferably, binding moieties areantibodies.

“Cell lines” refers to cells that have been separated from theiroriginal tissue, clonally multiplied and/or maintained in culture. Asspecific examples, cell lines may be derived from each type of cancerand multiple different cell lines may be derived from samples of thesame type of cancer. Examples of different types of cell lines include,but are not limited to, breast cancer cell lines, such as MCF-7,MDA-MB-453, MDA-MB-468, or T-47D or cell lines derived from othertissues, such as SKOV3 or HEK293.

“Chemotherapeutic agent” means a chemical substance that is used totreat a condition, particularly cancer.

A “cleavable linkage,” as used herein, refers to a chemical linkinggroup that may be cleaved to release a detectable molecular tagconnected to a binding moiety with the cleavable linkage.

A “cleavage-inducing moiety,” or “cleaving agent,” as used herein, is agroup that produces an active species that is capable of cleaving acleavable linkage. Preferably, the active species is a chemical speciesthat exhibits short-lived activity so that its cleavage-inducing effectsare only in the proximity of the site of its generation.

A “cleaving probe,” as used herein, refers to a reagent that comprises acleavage-inducing moiety, as defined herein, and a binding compound suchas an antibody, a peptide, a peptide or non-peptide ligand for a cellsurface receptor, a protein, such as streptavidin, a small molecule,such as biotin, an oligonucleotide, an oligonucleotide analog, such as apeptide nucleic acid, a lectin or any other molecular entity that iscapable of binding to a target protein or molecule or stable molecularcomplex.

“Dual kinase inhibitor” refers to molecules that inhibit more than onekinase, for example but not limited to, an inhibitor of both EGFR andHer-2 kinase activity, such as lapatinib.

“Effective proximity,” as used herein, describes the distance betweentwo binding compounds that is sufficient to generate a detectablesignal, indicating the presence of the target molecule. For example, aproximity probe and a binding compound that are bound on Her-3 (or withanother analyte of interest) within an effective proximity will generatea detectable signal, indicating and/or quantifying the presence of Her-3and/or a Her-3 complex. Preferably, the effective proximity range formany detection systems is less than 200 nM, more preferably, less than50 nM.

“EGFR”, “ErbB1”, “erbB-1”, “HER1”, “her-1”, “Her-1” and refers to theepidermal growth factor receptor and allelic variants thereof, asdescribed, for example, by Ono and Kuwano (see Ono and Kuwano (2006)Cuin. Cancer Res. 12:7242-7251) and Genbank accession number P00533.Unless indicated otherwise, the terms “EGFR”, “ErbB1”, “erbB-1”, “HER1”,“her-1”, “Her-1” when used herein refer to the human protein.

“Epitope” refers to a site on the surface of a molecule, usually aprotein, to which an antibody molecule or other binding compound binds.Generally, a protein has several or many different epitopes, also calledantigenic determinants, and reacts with antibodies of differentspecificities.

“FFPE” shall refer to a group of cells or quantity of tissue that arefixed, particularly conventional formalin-fixed paraffin-embeddedsamples. Such samples are typically, for example, without limitation,used in an assay for receptor complexes in the form of thin sections,e.g., 3-10 μm thick, of fixed tissue mounted on a microscope slide orequivalent surface. Such samples also typically undergo a conventionalre-hydration procedure, and optionally, an antigen retrieval procedureas a part of, or preliminary to, assay measurements.

As used herein, “greater than or equal to” (i.e., ≧ or >=) can incertain alternative embodiments mean “greater than” (>). Also, as usedherein, “less than or equal to” (i.e., ≦ or <=) can in certainalternative embodiments mean “less than” (<).

“Her-2”, “ErbB2”, “c-Erb-B2”, “HER2”, “Her2” and “neu” are usedinterchangeably herein and refer to native Her-2, and allelic variantsthereof, as described, for example, in Semba et al., 1985, Proc. Natl.Acad. Sci. USA 82:6497-650 and Yamamoto et al., 1986, Nature 319:230-234and Genbank accession number X03363. Unless indicated otherwise, theterms “Her-2”, “ErbB2”, “c-Erb-B2”, “HER2” and “Her2” when used hereinrefer to the human protein. The gene encoding Her2 is referred to hereinas “erbB2.”

“Her-2-acting agent,” as used herein, refers to a compound that canalter a biological activity of Her-2 or a Her-2 expressing cell or aHer-2 positive cancer cell. Such biological activities include, but arenot limited to, dimerization, autophosphorylation, phosphorylation ofanother receptor, signal transduction and the like. Biologicalactivities can include, without limitation, cell survival and cellproliferation and inhibition of such activities by a Her-2 acting agentcould be direct or indirect cell killing (e.g., ADCC), disruption ofprotein complexes or complex formation, modulation of proteintrafficking or enzyme inhibition. Biological activities can also includepatient response as set forth in this application. ExemplaryHer-2-acting agents include, but are not limited to, the large molecules4D5, pertuzumab, and trastuzumab and small molecules such as AEE-788 andlapatinib. A Her-2 complex is used to describe complexes of proteins,such as heterodimers, in which Her-2 is a component. A Her-2 complex mayinclude a Her-2 homodimer, or a heterodimer that includes Her-2 (e.g., aHer-2/Her-3 heterodimer).

“Her-3”, “ErbB3”, “c-erb-B3”, “erbB-3”, “HER3” and “Her3” are usedinterchangeably herein and refer to native Her-3, and allelic variantsthereof, as described, for example, in Kraus M H, et al. (1989) ProcNatl Acad Sci USA 86:9193-9197 and Plowman G D, et al. (1990) Proc NatlAcad Sci USA. 87:4905-4909 and Genbank accession number P21860. Unlessindicated otherwise, the terms “Her-3”, “ErbB3”, “c-erb-B3”, “erbB-3”,“HER3” and “Her3” when used herein refer to the human protein. The geneencoding Her3 is referred to herein as “erbB3.”

“Her-3 complex” is used to describe complexes of proteins, such asheterodimers, in which Her-3 is a component. Examples of heterodimersincluding Her-3 include but are not limited to Her-1/Her-3 andHer-2/Her-3.

“Her-3-targeted agent” or “Her-3 signaling pathway targeted agent”refers to therapeutics that alter the biological activity of Her-3 ormembers of the Her family signaling pathway. Such biological activitiesinclude, but are not limited to, dimerization, autophosphorylation,phosphorylation of another receptor, signal transduction and the like.Biological activities can include, without limitation, cell survival andcell proliferation and inhibition of such activities by a Her-3 or Her-3signaling pathway member acting agent could be direct or indirect cellkilling (e.g., ADCC), disruption of protein complexes or complexformation, modulation of protein trafficking or enzyme inhibition.Biological activities can also include patient response as set forth inthis application. Exemplary Her-3 or Her-3 signaling pathway memberacting agents might include, but are not limited to, large molecules(such as antibodies) or small molecules (such as small molecule kinaseinhibitors) targeted to Her-3, PI3K, Akt, mTOR, ERK1/2, or PYK2.

“High” refers to a measure that is greater than normal, greater than astandard such as a predetermined measure or a subgroup measure or thatis relatively greater than another subgroup measure. For example, highHer-3 refers to a measure of Her-3 that is greater than a normal Her-3measure. A normal Her-3 measure may be determined according to anymethod available to one skilled in the art. High Her-3 may also refer toa measure that is equal to or greater than a predetermined measure, suchas a predetermined cutoff. High Her-3 may also refer to a measure ofHer-3 wherein a high Her-3 subgroup has relatively greater levels ofHer-3 than another subgroup. For example, without limitation, accordingto the present specification, two distinct patient subgroups can becreated by dividing samples around a mathematically determined point,such as, without limitation, a median, thus creating a subgroup whosemeasure is high (ie, higher than the median) and another subgroup whosemeasure is low. Her-3 can be measured by any method known to one skilledin the art such as, for example, without limitation, using VERATAG® orusing any standard immunohistochemical (IHC) method. In some cases, a“high” expression level may comprise a range of expression that is veryhigh and a range of expression that is “moderately high” wheremoderately high is a level of expression that is greater than normal,but less than “very high”. Example ranges for high (including very highand moderately high) Her-2 expression and/or high Her-3 and/or high p95expression are provided herein.

“IHC, FISH and CISH” are methods (immunohistochemistry, fluorescence insitu hybridization, and chromogenic in situ hybridization, respectively)used to detect the presence of molecular entities in cells or tissues.For example, membrane receptors such as Her-3 and/or other members ofthe EGFR family of receptors can be detected using these methods.

“Isotype control antibodies” refers to antibodies that have the sameunderlying immunoglobulin structure as a specific antibody used as abinding compound but that do not have specificity for the targetedepitope. The use of isotype control antibodies allows one to observe anybinding that is due to non-specific binding.

“Likely to” (and “unlikely to”), as used herein, refers to an increased(or decreased) probability that an item, object, thing or person willoccur. Thus, in one example, a subject that is likely to respond totreatment with trastuzumab has an increased probability of responding totreatment with trastuzumab relative to a reference subject or group ofsubjects.

“Long,” as used herein, refers to a time measure that is greater thannormal, greater than a standard such as a predetermined measure or asubgroup measure that is relatively longer than another subgroupmeasure. For example, with respect to a patient's longevity, a long timeprogression refers to time progression that is longer than a normal timeprogression. Whether a time progression is long or not may be determinedaccording to any method available to one skilled in the art. In oneembodiment, “long” refers to a time that is greater than the median timecourse required for a significant event to occur in a disease.

“Low” is a term that refers to a measure that is less than normal, lessthan a standard such as a predetermined measure or a subgroup measurethat is relatively less than another subgroup measure. For example, lowHer-3 means a measure of Her-3 that is less than a normal Her-3 measurein a particular set of samples of patients. A normal Her-3 measure maybe determined according to any method available to one skilled in theart. Low Her-3 may also mean a measure that is less than a predeterminedmeasure, such as a predetermined cutoff. Low Her-3 may also mean ameasure wherein a low Her-3 subgroup is relatively lower than anothersubgroup. For example, without limitation, according to the presentspecification, two distinct patient subgroups can be created by dividingsamples around a mathematically determined point, such as, withoutlimitation, a median, thus creating a group whose measure is low (i.e.,less than the median) with respect to another group whose measure ishigh (i.e., greater than the median). Her-3 can be measured by anymethod known to one skilled in the art such as, for example, withoutlimitation, using the VERATAG® method or using any standardimmunohistochemical (IHC) method. Example ranges for low values ofHer-3, Her-2, and p95 expression are provided herein.

“Lysate” refers to the solution produced when the cell membranes ofcells are disrupted, whether by physical or chemical methods. Forexample, “tumor lysates” typically contain representative components ofthe cells comprising the tumor, including but not limited to, proteinmarkers, enzymes, nucleic acids and complexes of proteins and othermolecules that can subsequently be measured in various assays.

A “molecular tag,” as used herein, refers to a molecule that can bemeasured directly or indirectly, can be distinguished from othermolecules based on one or more physical, chemical or optical differencesamong the molecules being separated, including but not limited to,electrophoretic mobility, molecular weight, shape, solubility, pKa,hydrophobicity, charge, charge/mass ratio, polarity or the like. In oneembodiment, molecular tags in a plurality or set differ inelectrophoretic mobility and optical detection characteristics and canbe separated by electrophoresis. In another embodiment, molecular tagsin a plurality or set may differ in molecular weight, shape, solubility,pKa, hydrophobicity, charge, polarity and can be separated by normalphase or reverse phase HPLC, ion exchange HPLC, capillaryelectrochromatography, mass spectroscopy, gas phase chromatography or alike technique.

Measurement of molecular tags may also involve using secondary molecularinteractions, with or without further modification, to detect, enhanceor amplify a measurable signal that acts as a correlative for thepresence and/or quantity of an analyte, such as Her-3 or a Her-3 in acomplex. In one embodiment, a set of two or more molecular tags mayinteract within an effective proximity to produce a measurable signal.As molecular tags, a measurable signal may be generated, for example, bydetection of two complementary nucleic acid sequences that willhybridize when the complementary sequences are within an effectiveproximity. Other examples that either generate a measurable signal orthat can be measured using detection methods know in the art include,but are not limited to, FRET, BRET, BiFC, LCI and QPCR.

“Overall survival” or “OS” refers to a time as measured from the startof treatment to death or censor. Censoring may come from a study end orchange in treatment. Overall survival can refer to a probability as, forexample, a probability when represented in a Kaplan-Meier plot of beingalive at a particular time, that time being the time between the startof the treatment to death or censor.

“Pre-determined cutoff” as used herein, refers to the value of apredetermined measure on subjects exhibiting certain attributes thatallow the best discrimination between two or more categories of anattribute. For example, a pre-determined cutoff that allows one todiscriminate between two categories such as high Her-3 expression andlow Her-3 expression for determining overall survival may be used.Pre-determined cutoffs may be used to separate the subjects with valueslower than or higher than the pre-determined cutoff to optimize theprediction model.

A “proximity probe,” as used herein, refers to a reagent that comprisesa moiety capable of acting within effective proximity to a molecular tagon a binding compound to generate a detectable signal and an antibody, apeptide, a peptide or non-peptide ligand for a cell surface receptor, aprotein, such as streptavidin, a small molecule, such as biotin, anoligonucleotide, an oligonucleotide analog, such as a peptide nucleicacid, a lectin or any other molecular entity that is capable ofspecifically binding to a target protein or molecule or stable complex.For example, a proximity probe comprised of a Her-3-targeted antibodywith a molecular tag may be capable of binding to Her-3 within aneffective proximity to one or more Her-3 binding compounds, or a bindingcompound of another protein of interest, that has one or more moleculartags attached. In one embodiment, a proximity probe comprises a bindingmolecule and a first nucleic acid and a binding molecule comprises anantibody and a second nucleic acid, wherein the first and second nucleicacids are complementary to each other and each is a predetermined lengthso that when the nucleic acids are within an effective proximity of oneanother, they hybridize. Hybridization may be measured by any methodknown to one skilled in the art. For example, fluorophores may beattached to the nucleic acids as indicators of hybridization. In apreferred embodiment, hybridization is measured with a nucleic acidamplification method such as, for example, without limitation, therolling circle amplification method (see, for example, Lizardi et al.,(1998) Nat. Genet. 19: 225-232).

“RECIST” shall mean “Response Evaluation Criteria in Solid Tumours” andis a set of published rules that define when cancer patients improve(“respond”), stay the same (“stable”) or worsen (“progression”) duringtreatments. Response as defined by RECIST criteria have been published,for example, at Therasse et al., J. Natl. Canc. Inst. 92(3), Feb. 2,2000, and RECIST criteria may include other similar publisheddefinitions and rule sets.

“Respond” to treatment, and other forms of this verb, as used herein,refers to the reaction of a subject to treatment with an agent. As anexample, a subject responds to treatment if growth of a tumor in thesubject is retarded about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% ormore. In another example, a subject responds to treatment if a tumor inthe subject shrinks by about 5%, 10%, 20%, 30%, 40%, 50% or more asdetermined by any appropriate measure, e.g., by mass or volume. Inanother example, a subject responds to treatment with a Her2-actingagent if the subject experiences a life expectancy extended by about 5%,10%, 20%, 30%, 40%, 50% or more beyond the life expectancy predicted ifno treatment is administered. In another example, a subject responds totreatment with an agent if the subject has an overall survival orincreased time to progression. Several methods may be used to determineif a patient responds to a treatment including the RECIST criteria, asset forth herein.

“Sample” or “tissue sample” or “patient sample” or “patient cell ortissue sample” or “specimen” each refer to a collection of similar cellsobtained from a tissue of a subject or patient. The source of the tissuesample may be solid tissue as from a fresh tissue, frozen and/orpreserved organ or tissue or biopsy or aspirate; blood or any bloodconstituents, bodily fluids such as cerebral spinal fluid, amnioticfluid, peritoneal fluid or interstitial fluid or cells from any time ingestation or development of the subject. The tissue sample may containcompounds that are not naturally intermixed with the tissue in naturesuch as preservatives, anticoagulants, buffers, fixatives, nutrients,antibiotics or the like. Cells may be fixed in a conventional manner,such as in an FFPE manner.

“Short,” as used herein, refers to a time measure that is shorter thannormal, shorter than a standard such as a predetermined measure or asubgroup measure that is relatively shorter than another subgroupmeasure. For example, with respect to a patient's longevity, a shorttime progression refers to time progression that is shorter than anormal time progression or shorter than predicted. Whether a timeprogression is short or not may be determined according to any methodavailable to one skilled in the art. In one embodiment, “short” refersto a time that is less than the median time course required for asignificant event to occur in a disease.

“Signaling pathway”, as used herein, refers to a process in which thebinding of extracellular signaling molecules to cell-surface receptorstrigger events inside the cell and/or a process in which intracellularsignaling cascades can be triggered through intracellular interactions.For example, receptor tyrosine kinases are transmembrane proteins thatpropagate growth factor signals from the cell surface to intracellularprocesses that control critical functions such as growth,differentiation, angiogenesis and inhibition of apoptosis. In cancer,these signaling pathways are often exploited to facilitate tumor growthand metastasis. One such family of receptor tyrosine kinases is theepidermal growth factor receptor (EGFR) family. EGFR family members,EGFR, HER2, HER3 and HER4, are over-expressed in a wide variety of tumortypes.

“Significant event,” as used herein, shall refer to an event in apatient's disease that is important as determined by one skilled in theart. Examples of significant events include, for example, withoutlimitation, primary diagnosis, death, recurrence, the determination thata patient's disease is metastatic, relapse of a patient's disease or theprogression of a patient's disease from any one of the above notedstages to another. A significant event may be any important event usedto assess OS, TTP and/or using the RECIST or other response criteria, asdetermined by one skilled in the art.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, the terms “subject” and “subjects”refer to an animal, preferably a mammal including a non-primate (e.g., acow, pig, horse, donkey, goat, camel, cat, dog, guinea pig, rat, mouseor sheep) and a primate (e.g., a monkey, such as a cynomolgus monkey,gorilla, chimpanzee or a human).

“Targeted therapy” refers to therapeutic treatment that attempts toidentify and treat specific cells involved in disease without harming oraltering normal cells. Targeted therapeutics may be comprised of, butnot limited to, small molecules, such as lapatinib, gefitinib (Iressa®)and imatinib (Gleevec®), monoclonal antibodies, such as trastuzumab, ornucleic acids, such as siRNAs used to block expression of gene productsinvolved in disease processes. Targeted therapies are useful in thetreatment of many disease processes, such as cancer.

As used herein, “time course” shall refer to the amount of time betweenan initial event and a subsequent event. For example, with respect to apatient's cancer, time course may relate to a patient's disease and maybe measured by gauging significant events in the course of the disease,wherein the first event may be diagnosis and the subsequent event may bemetastasis, for example.

“Time to progression” or “TTP” refers to a time as measured from thestart of the treatment to progression or a cancer or censor. Censoringmay come from a study end or from a change in treatment. Time toprogression can also be represented as a probability as, for example, ina Kaplan-Meier plot where time to progression may represent theprobability of being progression free over a particular time, that timebeing the time between the start of the treatment to progression orcensor.

“Treatment,” and other forms of this word refer to the administration ofan agent to impede a disease, such as the growth of a cancer, to cause acancer to shrink by weight or volume, to extend the expected survivaltime of the subject and/or time to progression of the tumor or the like.Treatment may also refer to any course which one skilled, for example, atreating physician, deems expedient.

The term “VERATAG®” refers to single and multiplexed and multi-labelassays, materials, methods and techniques for performing and utilizingsuch assays, including but not limited to reagents, analyticalprocedures and software related to those assays. The terms VERATAG®,vTag® and ETAG® shall be used interchangeably.

In a first aspect, the invention is drawn to a method of measuringand/or quantifying the presence and/or amount of Her-3 and/or Her-3 in acomplex in a sample, the method comprising providing a sample anddetermining the presence and/or quantity of Her-3 and/or Her-3 in acomplex in the sample. In a preferred embodiment, the sample is abiological sample. In a preferred embodiment, the sample is a tissuesample. In a preferred embodiment, the sample is a fresh tissue sample,a fixed sample, a frozen sample or a lysate. In a preferred embodiment,the sample is a tumor sample. In a preferred embodiment, the sample is afrozen tumor tissue sample. In a preferred embodiment, the samplecomprises a tumor lysate from a fresh or frozen tumor sample. In apreferred embodiment, the sample is an FFPE sample or solubilized FFPEsample. In a preferred embodiment, the sample comprises a breast cancersample. In a certain embodiments, the breast cancer is early stage(i.e., adjuvant) breast cancer or metastatic breast cancer. In certainembodiments, the level of Her-2 expression in the breast cancer is high.In certain embodiments, high Her-2 expression is a log 10H2T≧ about1.14-1.25. In certain embodiments, the high Her-2 expression comprisesexpression that is very high and/or moderately high. In certainembodiments, the very high Her-2 expression is a log 10H2T≧ about1.84-2.21. Or, other ranges may be used depending upon the patientcohort. In certain embodiments, if the level of Her-3 is high, thepatient is less likely or unlikely to respond to the targeted therapy.In certain embodiments, if the level of Her-3 is low, the patient ismore likely to respond to the targeted therapy. In certain embodiments,as described in more detail herein, the therapy is a Her acting agent.In further embodiments, the therapy is at least one of a Her-2 actingagent or a Her-3-targeted agent.

In a preferred embodiment, the sample is a blood, plasma or lymphsample. In a preferred embodiment, the blood or plasma sample containscirculating tumor cells. In a preferred embodiment, the sample comprisescell lines. In a preferred embodiment, the measurement may bequantitative across a wide dynamic range.

In a preferred embodiment, the method comprises mixing the sample with abinding compound and determining the presence and/or quantity of thebinding compound bound to Her-3 and/or Her-3 in a complex. In apreferred embodiment, the binding compound binds specifically to Her-3.In a preferred embodiment, the binding compound comprises an antibody.In a preferred embodiment, the antibody is raised against one of thepeptides having SEQ ID NOs:1-8, as set forth in Example 2 and shown inFIG. 2A. In a preferred embodiment, the antibody is a monoclonalantibody comprising (a) a light chain variable region comprising CDR1,CDR2 and CDR3 having sequences as set forth in SEQ ID NOs:13, 14 and 15,respectively, and (b) a heavy chain variable region comprising CDR1,CDR2 and CDR3 having sequences as set forth in SEQ ID NOs:16, 17 and 18,respectively; and/or a monoclonal antibody comprising (a) a light chainvariable region comprising CDR1, CDR2 and CDR3 having sequences as setforth in SEQ ID NOs:19, 20 and 21, respectively, and (b) a heavy chainvariable region comprising CDR1, CDR2 and CDR3 having sequences as setforth in SEQ ID NOs:22, 23 and 24, respectively, as shown in Table 1B.In a preferred embodiment, the antibody is the antibody with the aminoacid sequence having SEQ ID NOs:9 and 11 as set forth in Table 1A forthe light and heavy chains, respectively, and/or SEQ ID NOs:10 and 12 asset forth in Table 1B for the light and heavy chains, respectively.

TABLE 1A Her3 light chain sequences: >Her3.B9A11.H1_LC (clone 6-6)SEQ ID NO: 9 MDSQAQVLILLLLWVSGTCGDIVMSQSPSSLAVSAGEKVTLSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPD RFTGSGSGTDFTLTVSSVQAEDLAVYYCKQSYNLWTFGGGTKLEIK >Her3.F9B10.3_LC (clone 7-3)SEQ ID NO: 10 MRCLAEFLGLLVLWIPGAIGDIVMTQGAPSVPVTPGESVSISCRSSKSLLQNNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPLTFGAGTKLGLKHer3 heavy chain sequences: >Her3.B9A11.H1_HC (clone 1-14) SEQ ID NO: 11MECNWILPFILSVTSGVYSEVQLQQPGTVLARPGASVRMSCKASGYTFTSYWMHWVKQRPGQGLEWIGAIYPGNSDTRDNQKFKGKAELTAVTSASTAYMELSSLTNEDSAVYYCTSYYFDGAGYFDFWGQGTTLTVSS >Her3.F9B10.3_HC (clone 2-1)SEQ ID NO: 12 MEWSWVFLFLLSVIASVQSQVQLQQSGAEVVRPGASVTLSCKASAYTFTDYELHWMRQTPVHGLEWIGASDPETGGSAYNQKFKGKAILTADKSSSTAFMELRSLTSEDSAVYFCTRRIFYFGSRGDFFDYWGQGTSLTVSS

TABLE 1B Complementarity Determining Regions (CDRs) Her3.B9A11.H1_LCHer3.B9A11.H1_HC Her3.F9B10.3_LC Her3.F9B10.3_HC CDR1 KSSQSLLNSRTRKNYLASYWMH RSSKSLLQNNGNTYLY DYELH SEQ ID NO: 13 SEQ ID NO: 16 SEQ ID NO: 19SEQ ID NO: 22 CDR2 WASTRES AIYPGNSDTRDNQKFKG RMSNLAS ASDPETGGSAYNQKFKGSEQ ID NO: 14 SEQ ID NO: 17 SEQ ID NO: 20 SEQ ID NO: 23 CDR3 KQSYNLWTYYFDGAGYFDF MQHLEYPLT RIFYFGSRGDFFDY SEQ ID NO: 15 SEQ ID NO: 18SEQ ID NO: 21 SEQ ID NO: 24TABLE 1A and 1B. Table 1A shows the amino acid sequences of the lightand heavy chains of two antibodies that bind Her3, Her3.B9A11.H1 andHer3.F9B10.3. The clonal isolates from which the sequences were derivedare shown and the complementarity-determining regions (CDRs) areunderlined. The light and heavy chains are denoted by “_LC” or “_HC”,respectively. Table 1B shows the three CDRs for the B9A11.H1 light andheavy chains and the F9B10.3 light and heavy chains, respectively.

In a preferred embodiment, the method comprises mixing (i) a sample thatmay contain Her-3 and/or Her-3 in a complex; (ii) a proximity probe thatis capable of binding Her-3 and/or at least one other analyte, theproximity probe having an effective proximity and (iii) at least onebinding compound, the at least one binding compound being capable ofbinding Her-3 and having one or more signaling molecules attached,wherein binding of the proximity probe and the binding compound withinthe effective proximity produces a signal from the molecular tags thatcorrelates with the presence and/or quantity of Her-3 and/or Her-3 in acomplex. In a preferred embodiment, the proximity probe and/or bindingcompound is capable of binding specifically to Her-3. In a preferredembodiment, the proximity probe and/or binding compound furthercomprises an antibody and each antibody may bind to a specific epitopeon Her-3. In a preferred embodiment, the antibody is raised against oneof the peptides having SEQ ID NOs:1-8, as set forth in Example 2 andshown in FIG. 2A. In a preferred embodiment, the antibody is amonoclonal antibody comprising (a) a light chain variable regioncomprising CDR1, CDR2 and CDR3 having sequences as set forth in SEQ IDNOs:13, 14 and 15, respectively, and (b) a heavy chain variable regioncomprising CDR1, CDR2 and CDR3 having sequences as set forth in SEQ IDNOs:16, 17 and 18, respectively; and/or a monoclonal antibody comprising(a) a light chain variable region comprising CDR1, CDR2 and CDR3 havingsequences as set forth in SEQ ID NOs:19, 20 and 21, respectively, and(b) a heavy chain variable region comprising CDR1, CDR2 and CDR3 havingsequences as set forth in SEQ ID NOs:22, 23 and 24, respectively (Table1B). In a preferred embodiment, the antibody is the antibody with theamino acid sequence having SEQ ID NOs:9 and 11 as set forth in Table 1Afor the light and heavy chains, respectively, and/or SEQ ID NOs:10 and12 as set forth in Table 1A for the light and heavy chains,respectively. In a preferred embodiment, the sample is a biologicalsample. In a preferred embodiment, the sample is a tissue sample. In apreferred embodiment, the sample is a fixed sample, a frozen sample or alysate. In a preferred embodiment, the sample is a tumor sample. In apreferred embodiment, the sample is a frozen tumor tissue sample. In apreferred embodiment, the sample comprises a tumor lysate. In apreferred embodiment, the sample comprises a breast cancer sample asdiscussed herein. For example, in a certain embodiments, the breastcancer is early stage (i.e., adjuvant) breast cancer or metastaticbreast cancer. In certain embodiments, the level of Her-2 expression inthe breast cancer is high. In certain embodiments, high Her-2 expressionis a log 10H2T≧ about 1.14-1.25. In certain embodiments, the high Her-2expression comprises expression that is very high and/or moderatelyhigh. In certain embodiments, the very high Her-2 expression is a log10H2T≧ about 1.84-2.21. Or, other ranges may be used depending upon thepatient cohort.

In a preferred embodiment, the sample is an FFPE sample or solubilizedFFPE sample. In a preferred embodiment, the sample is a blood, plasma orlymph sample. In a preferred embodiment, the blood or plasma samplecontains circulating tumor cells. In a preferred embodiment, the samplecontains exosomes and/or other vesicles. In a preferred embodiment, thesample comprises cell lines. In a preferred embodiment, the measurementmay be quantitative across a wide dynamic range. In a preferredembodiment, the wide dynamic range is approximately 2 logs. In a morepreferred embodiment, the wide dynamic range is about 1-1.5 logs inbreast cancer samples. In a preferred embodiment, the method provides aquantitative continuum of Her-3 expression. In a preferred embodiment,the measurement or quantity is sensitive to at least about 1000receptors per cell to about 200,000 receptors per cell as determined byaccuracy studies utilizing well-characterized cell line models andcross-validating technologies such as ELISA and flow cytometry. In apreferred embodiment, the measurement or quantity is sensitive to atleast about 5000 receptors per cell to about 200,000 receptors per cell.In a preferred embodiment, the measurement or quantity is sensitive toat least about 10,000 receptors per cell to about 200,000 receptors percell. In a preferred embodiment, the measurement or quantity issensitive to at least about 25,000 receptors per cell to about 200,000receptors per cell. In a preferred embodiment, the measurement isspecific as determined using isotype control antibodies and comparisonwith conventional IHC methods.

In a preferred embodiment, determining the presence and/or quantity ofbinding compound bound to Her-3 further comprises providing a secondbinding compound, the second binding compound being able to specificallybind the binding compound bound to Her-3 and determining the presenceand/or quantity of the second binding compound as correlative of thepresence and/or quantity of the binding compound bound to Her-3. In apreferred embodiment, the second binding compound is an antibody.

The use of a second binding compound that is capable of specificallybinding the first binding compound and has one or more molecular tagsmay have practical advantages. For example, multiple Her-3-specificfirst binding compounds may be tested using a single second bindingcompound to which is attached one or more molecular tags, abrogating theneed for attaching molecular tags to each of the multiple Her-3-specificfirst binding compounds. In a preferred embodiment, the first bindingcompound is a mouse antibody and the second binding compound is ananti-mouse antibody raised in a non-mouse species (e.g., goat anti-mouseantibodies) to which cleavable molecular tags have been attached.

Second binding compounds are typically labeled with probes useful fordetection. Detection systems commonly used for detecting second bindingcompounds include but are not limited to cleavable molecular tags, asdescribed herein; radiolabels (i.e., radioisotopes such as I-125);enzymes that convert a chemical into a measurable colorimetric,fluorescent or electrochemical signal (e.g., peroxidases) andfluorescent proteins (e.g., green fluorescent protein and its manyderivatives).

The antibody can be monoclonal, polyclonal or recombinant and can beprepared by techniques that are well known in the art. Antibodies mayinclude a complete immunoglobulin or fragment thereof, whichimmunoglobulins include the various classes and isotypes, such as IgA,IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereof mayinclude Fab, Fv and F(ab′)2, Fab′ and the like. Antibodies may also besingle-chain antibodies, chimeric antibodies, humanized antibodies orany other antibody derivative known to one of skill in the art thatretains binding activity that is specific for a particular binding site.In addition, aggregates, polymers and conjugates of immunoglobulins ortheir fragments can be used where appropriate so long as bindingaffinity is maintained.

To facilitate the development of methods to measure Her-3 in biologicalsamples, Her-3-specific monoclonal antibodies were created. Mice wereimmunized against peptides from Her-3 (shown in FIG. 2A) and standardmethods as set forth further herein and as known to one skilled in theart were used to create hybridomas. Many methods are known for thecreation and production of monoclonal antibodies, for example, thehybridoma method as first described by Koehler et al. (1975) Nature256:495-497 or other methods described in the literature (see Goding, JW (1980) J. Immunol. Methods 34:285-308; Harlow E and Lane D (1988) inAntibodies: A Laboratory Manual, Chapter 6; Kennett R H et al. (1980)Monoclonal Antibodies, Plenum Press; Zola H (1987) MonoclonalAntibodies: A Manual of Techniques, CRC Press).

In one embodiment, the method of creating hybridomas begins withimmunizing a host animal, such as a mouse, to elicit the production oflymphocytes that produce antibodies targeted to the peptide orprotein(s) of interest. Lymphocytes may also be immunized in vitro. Theantigen used may be a peptide, a protein or a cell displaying theantigen on the cell surface. Lymphocytes are collected then fused bychemical (e.g., with PEG) or electrical (e.g., by electrofusion) methodswith myeloma cells to form hybridoma cells, typically under conditionsthat prevent the growth and/or survival of the parent myeloma cells.Fused cells are allowed to grow because they contain enzymes thatfacilitate survival in the culture medium. In a preferred embodiment,the culture medium contains hypoxanthine, aminopterin and thymidine (HATmedium), which prevents the growth of cells lacking hypoxanthine quininephosphoribosyl transferase (HPRT). The HPRT is supplied to the fusedcell by the lymphocyte partner, allowing survival of the hybridoma butpreventing survival of the parent myeloma cells, which lack HPRT.

Culture media in which hybridomas are grown (i.e., conditioned media)are typically assayed for the production of monoclonal antibodiesdirected against the antigen using a variety of techniques (see Voller,et al. (1978) J. Clin. Pathol. 31:507-520), including but not limitedto, immunoprecipitation or an in vitro binding assay such asenzyme-linked immunosorbant assay (ELISA; see Engvall E (1977) inBiomedical Applications of Immobilized Enzymes and Proteins, edited byTMS Chang, 2:87-96, Plenum Press), radioimmunoassay (MA; see Sonksen P H(1974) Brit. Med. Bull. 30:1-103), Western blots or flow cytometry.Conditioned media from the hybridomas were profiled in a series ofassays including ELISA (FIG. 1), Western blot (FIG. 2) and flowcytometry (FIG. 3). In preferred embodiments, studies using both nativeand permeabilized and fixed cells are performed to identify antibodiesthat may perform well in applications that use fixed cells or tissues,such as immunohistochemistry (IHC). Clones of interest may be subclonedby limiting dilution or single cell flow cytometry.

As will be known to those skilled in the art, monoclonal antibodiessecreted by hybridoma clones (or subclones) can be purified usingconventional purification procedures such as, but not limited to,dialysis, affinity chromatography, gel electrophoresis or proteinA-sepharose (or protein L-agarose) chromatography.

One antibody generated, B9A11 (i.e., SEQ ID NOs:9 and 11), which wasraised against the peptide CFDNPDYWHSRLFPKANA (SEQ ID NO:6) from Her-3,was chosen for use in the Her3− VERATAG® assay experiments describedherein. The hybridoma cell lines that produce antibodies B9A11 and F9B10were deposited with the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110, on Jan. 12, 2010, under theterms of the Budapest Treaty and were accorded the ATCC accessionnumbers PTA-10574 and PTA-10575.

Many methods and reagents are commonly used to prepare biologicalsamples for analysis. Several methods are outlined or referenced hereinand many others are known to those skilled in the art. Samplescontaining Her-3 suitable for use as biomarkers may come from a widevariety of sources, including cell cultures, animal or plant tissues,patient biopsies, blood or the like. Preferably, samples are humanpatient samples. Samples are prepared for assays of the invention usingconventional techniques, which may depend on the source from which asample is taken. For biopsies and medical specimens, guidance isprovided in the following references: Bancroft J D & Stevens A, eds.,1977, Theory and Practice of Histological Techniques, ChurchillLivingstone, Edinburgh; Pearse, 1980, Histochemistry. Theory andapplied, 4^(th) ed., Churchill Livingstone, Edinburgh.

Examples of patient tissue samples that may be used include, but are notlimited to tissues of breast, prostate, ovary, colon, lung, endometrium,stomach, salivary gland or pancreas. The tissue sample can be obtainedby a variety of procedures including surgical excision, aspiration orbiopsy. The tissue may be fresh or frozen. In one embodiment, thebiological sample may be cells cultured in vitro and collected bycentrifugation as a cell pellet. In one embodiment, the samples may bepatient blood samples or specific blood cell types or subsets of bloodcell types (e.g., buffy coats). In one embodiment, the biological samplemay be exosomes or samples containing exosomes. Exosomes are small(30-200 nm) vesicles that can be secreted by most cell types, includingtumor cells (see Mignot et al (2006) J. Cell. Mol. Med. 10:376-3 88), invivo and in vitro. Tumor-derived exosomes are thought to play a role inthe ability of tumors to evade the immune system and have potential forboth diagnostic and therapeutic applications (see Taylor and Black(1985) J. Natl. Cancer Inst. 74:859-867) and are therefore biologicalsamples of interest.

In a preferred embodiment, the sample is a tumor sample. Examples oftypes of tumor samples include cancers such as, without limitation,carcinomas, sarcomas, myelomas, leukemias, lymphomas and mixed typecancers. In one embodiment, the cancer is a bone cancer, for example,Ewing's sarcoma, osteosarcoma and rhabdomyosarcoma and other soft-tissuesarcomas. In another embodiment, the cancer is a brain tumor, forexample, oligodendroglioma, ependymoma, menengioma, lymphoma, schwannomaor medulloblastoma. In another embodiment, the cancer is breast cancer.In another embodiment, the cancer is an endocrine system cancer, forexample, adrenal, pancreatic, parathyroid, pituitary and thyroidcancers. In another embodiment, the cancer is a gastrointestinal cancer,for example, anal, colorectal, esophageal, gall bladder, gastric, liverand small intestine cancers. In another embodiment, the cancer is agynecological cancer, for example, cervical, endometrial, uterine,fallopian tube, gestational trophoblastic disease, choriocarcinoma,ovarian, vaginal or vulvar cancer. In another embodiment, the cancer isa head and neck cancer, for example, laryngeal, oropharyngeal,parathyroid or thyroid cancer. In another embodiment, the cancer ismelanoma, squamous cell carcinoma or basal cell carcinoma. In anotherembodiment, the cancer is a leukemic cancer, for example, acutelymphocytic leukemia, acute myelogenous leukemia, chronic lymphocyticleukemia, chronic myelogenous leukemia, hairy cell leukemia or amyeloproliferative disorder. In another embodiment, the cancer is a lungcancer, for example, a mesothelioma or non-small cell lung cancer. Inanother embodiment, the cancer is a lymphoma, such as cutaneous T celllymphoma, Hodgkin's disease or non-Hodgkin's disease. In anotherembodiment, the cancer is metastatic cancer. In another embodiment, thecancer is a myeloma, for example, a multiple myeloma. In anotherembodiment, the cancer is penile cancer. In another embodiment, thecancer is prostate cancer. In another embodiment, the cancer istesticular cancer. In another embodiment, the cancer is thyroid cancer,for example, papillary, follicular, medullary or anaplastic orundifferentiated thyroid carcinoma. In another embodiment, the cancer isa urinary tract cancer, for example, bladder, kidney or urethral cancer.

Methods for preparing cells cultured in vitro as fresh, frozen or fixedsamples are known to those with skill in the art and exemplary methodsare described herein. In one embodiment, assays of the invention arecarried out on tissue samples that have been fixed and embedded inparaffin and a step of deparaffination may be carried out. A tissuesample may be fixed (i.e., preserved) by conventional methodology. See,e.g., Lee G. Luna, HT (ASCP) ed., 1960, Manual of Histological StainingMethod of the Armed Forces Institute of Pathology 3rd edition, TheBlakiston Division McGraw-Hill Book Company, New York; Ulreka V. Mikel,ed., 1994, The Armed Forces Institute of Pathology Advanced LaboratoryMethods in Histology and Pathology, Armed Forces Institute of Pathology,American Registry of Pathology, Washington, D.C. One of skill in the artwill appreciate that the choice of a fixative is determined by thepurpose for which the tissue is to be histologically stained orotherwise analyzed. One of skill in the art will also appreciate thatthe length of fixation depends upon the size of the tissue sample andthe fixative used.

Generally, a tissue sample is first fixed and is then dehydrated throughan ascending series of alcohols, infiltrated and embedded with paraffinor other sectioning media so that the tissue sample may be sectioned.Alternatively, one may section the tissue and fix the sections obtained.By way of example, the tissue sample may be embedded and processed inparaffin by conventional methodology according to conventionaltechniques or as described herein. Once the tissue sample is embedded,the sample may be sectioned by a microtome according to conventionaltechniques. Sections may have a thickness in a range from about threemicrons to about twelve microns, and preferably, a thickness from about5 microns to about 10 microns. In one embodiment, a section may have anarea from about 10 mm² to about 1 cm². Once cut, the sections may beattached to slides by several standard methods. Examples of slideadhesives include, but are not limited to, silane, gelatin andpoly-L-lysine. Paraffin-embedded sections may be attached to positivelycharged slides and/or slides coated with poly-L-lysine.

If paraffin has been used as the embedding material, the tissue sectionsare generally deparaffinized and rehydrated prior to detection ofbiomarkers. Tissue sections may be deparaffinized by severalconventional standard methodologies. For example, xylenes and agradually descending series of alcohols may be used according toconventional techniques described by the references provided herein.Alternatively, commercially available deparaffinizing non-organic agentssuch as Hemo-De® (CMS, Houston, Tex.) may be used.

Cell lysates of mammalian tissue culture cells or fresh or frozentissues may be prepared by conventional cell lysis techniques (e.g.,0.14 M NaCl, 1.5 mM MgCl₂, 10 mM Tris-HCl (pH 8.6), 0.5% Nonidet P-40,and protease and/or phosphatase inhibitors as required). For freshmammalian tissues, sample preparation may also include a tissuedisaggregation step, such as crushing, mincing, grinding or sonication.

Stable cell lines expressing varying levels of Her-3 were generated.Cell lines stably expressing varying levels of a protein of interest areuseful in validating new assays, such as the Her-3 VERATAG® assay, withrespect to many parameters such as optimal antibody concentrations,accuracy, sensitivity, reproducibility, precision, linearity,specificity and dynamic range. HEK 293 cells were used to create thestable Her-3-expressing cell lines. HEK 293 cells are a specific cellline originally derived from human embryonic kidney cells transformedwith adenovirus DNA (see Graham et al. (1977) J. Gen. Virol. 36:59-74).HEK 293 cells are easy to grow in culture, transfect readily and havebeen widely used in cell biology research as well as in proteinproduction for the biotechnology industry for many years. The generationof the Her-3− expressing cell lines is described in Example 1 and theresults of ELISA assays to determine the level of Her-3 in each of thesecell lines is shown in FIG. 1.

In a further preferred embodiment, the proximity probe comprises anantibody and a first nucleic acid and the binding compound comprises anantibody and a second nucleic acid, wherein the first and the secondnucleic acids are complementary to each other and able to hybridize todetermine the effective proximity and produce the signal, directly orindirectly, through hybridization. In a preferred embodiment, theproximity probe and/or binding compound is capable of bindingspecifically to Her-3. In a preferred embodiment, the binding compoundand/or the proximity probe further comprises an antibody and eachantibody binds to a different epitope on Her-3. In a preferredembodiment, the antibody is raised against one of the peptides havingSEQ ID NOs:1-8, as set forth in Example 2 and shown in FIG. 2A. In apreferred embodiment, the antibody is a monoclonal antibody comprising(a) a light chain variable region comprising CDR1, CDR2 and CDR3 havingsequences as set forth in SEQ ID NOs:13, 14 and 15, respectively, and(b) a heavy chain variable region comprising CDR1, CDR2 and CDR3 havingsequences as set forth in SEQ ID NOs:16, 17 and 18, respectively; and/ora monoclonal antibody comprising (a) a light chain variable regioncomprising CDR1, CDR2 and CDR3 having sequences as set forth in SEQ IDNOs:19, 20 and 21, respectively, and (b) a heavy chain variable regioncomprising CDR1, CDR2 and CDR3 having sequences as set forth in SEQ IDNOs:22, 23 and 24, respectively (Table 1B). In a preferred embodiment,the antibody is the antibody with the amino acid sequence having SEQ IDNOs:9 and 11 as set forth in Table 1A for the light and heavy chains,respectively, and/or SEQ ID NOs:10 and 12 as set forth in Table 1A forthe light and heavy chains, respectively. In a preferred embodiment, thesample is a biological sample. In a preferred embodiment, the sample isa tissue sample. In a preferred embodiment, the sample is a fixedsample, a frozen sample or a lysate. In a preferred embodiment, thesample is a tumor sample. In a preferred embodiment, the sample is afrozen tumor tissue sample. In a preferred embodiment, the samplecomprises a tumor lysate. In a preferred embodiment, the samplecomprises a breast cancer sample as described herein. In a preferredembodiment, the sample is an FFPE sample or solubilized FFPE sample. Ina preferred embodiment, the sample is a blood, plasma or lymph sample.In a preferred embodiment, the blood or plasma sample containscirculating tumor cells. In a preferred embodiment, the sample containsexosomes and/or other vesicles. In a preferred embodiment, the samplecomprises cell lines. In a preferred embodiment, the measurement may bequantitative across a wide dynamic range. In a preferred embodiment, thewide dynamic range is about 2 logs. In a more preferred embodiment, thewide dynamic range is about 1-1.5 logs in breast cancer samples. In apreferred embodiment, the method provides a quantitative continuum ofHer-3 expression. In a preferred embodiment, the measurement or quantityis sensitive to at least about 1000 receptors per cell to about 200,000receptors per cell as determined by accuracy studies utilizingwell-characterized cell line models and cross-validating technologiessuch as ELISA and flow cytometry. In a preferred embodiment, themeasurement or quantity is sensitive to at least about 5000 receptorsper cell to about 200,000 receptors per cell. In a preferred embodiment,the measurement or quantity is sensitive to at least about 10,000receptors per cell to about 200,000 receptors per cell. In a preferredembodiment, the measurement or quantity is sensitive to at least about25,000 receptors per cell to about 200,000 receptors per cell. In apreferred embodiment, the measurement is specific as determined usingisotype control antibodies and comparison with conventional IHC methods.Examples of proximity probes and binding compounds, as set forth herein,can be found, for example, in U.S. Pat. Nos. 7,306,904; 7,320,860 and7,351,528, each of which is incorporated by reference herein, includingany drawings.

Proximity assays are increasingly useful for the understanding of thebiological role of molecular complexes, as well as in the study ofbiomarkers. For example, binding compounds that specifically bind Her-3or Her-3 in a complex can be coupled with many different detectionsystems to measure the presence and/or quantity of Her-3 or Her-3 in acomplex. Any method known to one of skill in the art to be useful fordetermining an amount of Her-3 or Her-3 in a complex can be used inaccordance with the present invention. Such methods include but are notlimited to Foerster resonance energy transfer (FRET), bioluminescenceresonance energy transfer (BRET), biomolecular fluoresencecomplementation, proximity ligation assay (PLA), scintillation proximityassay (SPA) and rolling circle amplification (RCA) or any other methodfor detecting nucleic acid duplexes formed by the proximity of a bindingprobe and a proximity probe with complementary strands of nucleic acids.

In conducting the methods of the invention, a combination of the assaycomponents is made, including the sample being tested, the bindingcompounds and optionally the proximity probe. Generally, assaycomponents may be combined in any order. In certain applications,however, the order of addition may be relevant. For example, one maywish to monitor competitive binding, such as in a quantitative assay. Orone may wish to monitor the stability of an assembled complex. In suchapplications, reactions may be assembled in stages.

The amounts of each reagent can generally be determined empirically. Theamount of sample used in an assay will be determined by the predictednumber of target complexes present and the means of separation anddetection used to monitor the signal of the assay. In general, theamounts of the binding compounds and the proximity probe can be providedin molar excess relative to the expected amount of the target moleculesin the sample, generally at a molar excess of at least about 1.5, moredesirably about 10-fold excess, or more. In specific applications, theconcentration used may be higher or lower, depending on the affinity ofthe binding compound or proximity probe and the expected number oftarget molecules present on a single cell.

The assay mixture can be combined and incubated under conditions thatprovide for binding of the probes to the cell surface molecules, usuallyin an aqueous medium, generally at a physiological pH (comparable to thepH at which the cells are cultured), maintained by a buffer at aconcentration in the range of about 10 to 200 mM. Conventional buffersmay be used, as well as other conventional additives as necessary, suchas salts, growth medium, stabilizers, etc. Physiological and constanttemperatures are normally employed. Incubation temperatures normallyrange from about 4° to 70° C., usually from about 15° to 45° C., moreusually about 25° to 37° C.

In a preferred embodiment, the proximity probe comprises a cleavingprobe that has a cleavage-inducing moiety and the at least one bindingcompound has one or more molecular tags attached to the binding compoundby a cleavable linkage, wherein the cleavable linkage may be cleavedwithin the effective proximity, producing a signal that correlates withthe presence and/or quantity of Her-3. In a preferred embodiment, thecleaving probe and/or binding compound is capable of bindingspecifically to Her-3. In a preferred embodiment, the binding compoundand/or the proximity probe further comprises an antibody and eachantibody binds to a different epitope on Her-3. In a preferredembodiment, the antibody is raised against one of the peptides havingSEQ ID NOs:1-8, as set forth in Example 2 and shown in FIG. 2A. In apreferred embodiment, the antibody is a monoclonal antibody comprising(a) a light chain variable region comprising CDR1, CDR2 and CDR3 havingsequences as set forth in SEQ ID NOs:13, 14 and 15, respectively, and(b) a heavy chain variable region comprising CDR1, CDR2 and CDR3 havingsequences as set forth in SEQ ID NOs:16, 17 and 18, respectively; and/ora monoclonal antibody comprising (a) a light chain variable regioncomprising CDR1, CDR2 and CDR3 having sequences as set forth in SEQ IDNOs:19, 20 and 21, respectively, and (b) a heavy chain variable regioncomprising CDR1, CDR2 and CDR3 having sequences as set forth in SEQ IDNOs:22, 23 and 24, respectively. In a preferred embodiment, the antibodyis the antibody with the amino acid sequence having SEQ ID NOs:9 and 11as set forth in Table 1A for the light and heavy chains, respectively,and/or SEQ ID NOs:10 and 12 as set forth in Table 1A for the light andheavy chains, respectively. In a preferred embodiment, the sample is abiological sample. In a preferred embodiment, the sample is a tissuesample. In a preferred embodiment, the sample is a fixed sample, afrozen sample or a lysate. In a preferred embodiment, the sample is atumor sample. In a preferred embodiment, the sample is a frozen tumortissue sample. In a preferred embodiment, the sample comprises a tumorlysate. In a preferred embodiment, the sample comprises a breast cancersample as described herein. In a preferred embodiment, the sample is anFFPE sample or solubilized FFPE sample. In a preferred embodiment, thesample is a blood, plasma or lymph sample. In a preferred embodiment,the blood or plasma sample contains circulating tumor cells. In apreferred embodiment, the sample comprises cell lines. In a preferredembodiment, the measurement may be quantitative across a wide dynamicrange. In a preferred embodiment, the wide dynamic range is about 2logs. In a more preferred embodiment, the wide dynamic range is about1-1.5 logs in breast cancer samples. In a preferred embodiment, themethod provides a quantitative continuum of Her-3 expression. In apreferred embodiment, the measurement or quantity is sensitive to atleast about 1000 receptors per cell to about 200,000 receptors per cellas determined by accuracy studies utilizing well-characterized cell linemodels and cross-validating technologies such as ELISA and flowcytometry. In a preferred embodiment, the measurement or quantity issensitive to at least about 5000 receptors per cell to about 200,000receptors per cell. In a preferred embodiment, the measurement orquantity is sensitive to at least about 10,000 receptors per cell toabout 200,000 receptors per cell. In a preferred embodiment, themeasurement or quantity is sensitive to at least about 25,000 receptorsper cell to about 200,000 receptors per cell. In a preferred embodiment,the measurement is specific as determined using isotype controlantibodies and comparison with conventional IHC methods.

A two antibody proximity assay was optimized. Ab-6 (LabVision), aHer-3-specific monoclonal antibody with epitope specificity to thecytoplasmic terminus of Her-3, was conjugated to the VERATAG® reporter(Pro 11) for use as the proximity probe (Ab-6-Pro11). The proprietarymonoclonal antibody, B9A11, was conjugated to biotin for use as thecleaving probe (B9A11-biotin) when complexed with streptavidin-methyleneblue (“molecular scissors”). The assay methods are described in Example5. Several cell lines that expressed varying levels of Her-3 rangingfrom very high levels (in one of the stably transfected HEK 293 celllines, called 293H3-Clone 1) to moderate to low levels (MDA-MB-468 andMDA-MB-453, respectively) to low to no detectable Her-3 (in SKOV3 cells)were used in the optimization studies. The optimization process includeddetermining the optimal antibody concentration for maximizing dynamicrange (see Example 7 and FIG. 6), determining the accuracy of the assay(see Example 8 and FIG. 7), testing the sensitivity, reproducibility,and precision of the assay (see Example 9/FIG. 8, Example 10/FIG. 9 andExample 11/FIG. 10, respectively), the linearity of the assay indifferent sample sizes (see Example 12 and FIG. 11) and the specificityof the assay by testing for non-specific binding using isotype controls(see Example 13 and FIG. 12).

Isotype controls are typically performed to eliminate the possibilitythat the binding results are due to the particular isotype of theantibody rather than the individual antibody. Additionally, one skilledin the art will appreciate that any signal “noise” seen in the isotypecontrols can be subtracted from the total signal, potentially yielding amore refined result. When an isotype control experiment was performed,the contribution of non-specific binding was observed to be very low(see FIG. 12).

Many advantages are provided by measuring Her-3 or the Her-3 in acomplex using releasable molecular tags, including separation ofreleased molecular tags from an assay mixture providing greatly reducedbackground and a significant gain in sensitivity and separation anddetection providing a convenient multiplexing capability so thatmultiple receptor complex components may be readily measuredsimultaneously in the same assay. Assays employing such tags can have avariety of forms and are disclosed in the following references: U.S.Pat. Nos. 7,105,308; 6,627,400; 7,402,397; 7,402,398 and 7,402,399, aswell as International Patent Publication No. WO 2004/011900, each ofwhich is incorporated herein by reference in its entirety. A widevariety of separation techniques may be employed that can distinguishmolecules based on one or more physical, chemical or optical differencesamong molecules being separated including electrophoretic mobility,molecular weight, shape, solubility, pKa, hydrophobicity, charge,charge/mass ratio or polarity. In one embodiment, molecular tags in aplurality or set differ in electrophoretic mobility and opticaldetection characteristics and are separated by electrophoresis. Inanother embodiment, molecular tags in a plurality or set may differ inmolecular weight, shape, solubility, pKa, hydrophobicity, charge,polarity and are separated by normal phase or reverse phase HPLC, ionexchange HPLC, capillary electrochromatography, mass spectroscopy or gasphase chromatography.

Sets of molecular tags are provided that can be separated into distinctbands or peaks by a separation technique after they are released frombinding compounds. Identification and quantification of such peaksprovides a measure or profile of the presence and/or amounts of Her-3.Molecular tags within a set may be chemically diverse; however, forconvenience, sets of molecular tags are usually chemically related. Forexample, they may all be peptides or they may consist of differentcombinations of the same basic building blocks or monomers or they maybe synthesized using the same basic scaffold with different substituentgroups for imparting different separation characteristics. The number ofmolecular tags in a plurality may vary depending on several factorsincluding the mode of separation employed, the labels used on themolecular tags for detection, the sensitivity of the binding moietiesand the efficiency with which the cleavable linkages are cleaved.

Measurements made directly on tissue samples may be normalized byincluding measurements on cellular or tissue targets that arerepresentative of the total cell number in the sample and/or the numbersof particular subtypes of cells in the sample (see, e.g., U.S. PatentApplication Publication No. US 2009/0191559, which is incorporated byreference herein in its entirety, including any drawings). Theadditional measurement may be preferred, or even necessary, because ofthe cellular and tissue heterogeneity in patient samples, particularlytumor samples, which may comprise substantial fractions of normal cells.

In one embodiment, a binding compound can be represented by thefollowing formula:

B-(L-E)_(k)

wherein B is binding moiety; L is a cleavable linkage and E is amolecular tag. In homogeneous assays, cleavable linkage, L, may be anoxidation-labile linkage, and more preferably, it is a linkage that maybe cleaved by singlet oxygen. The moiety “-(L-E)_(k)” indicates that asingle binding compound may have multiple molecular tags attached viacleavable linkages. In one aspect, k is an integer greater than or equalto one, but in other embodiments, k may be greater than several hundred,e.g., 100 to 500 or k is greater than several hundred to as many asseveral thousand, e.g., 500 to 5000. Usually each of the plurality ofdifferent types of binding compounds has a different molecular tag, E.Cleavable linkages, e.g., oxidation-labile linkages, and molecular tags,E, are attached to B by way of conventional chemistries.

Preferably, B is an antibody that specifically binds to a target, suchas Her-3. Antibodies specific for Her-3 epitopes are provided in theexamples set forth herein. Antibody compositions may be readily formedfrom a wide variety of commercially available antibodies, eithermonoclonal or polyclonal or by methods disclosed herein.

Cleavable linkage, L, can be virtually any chemical linking group thatmay be cleaved under conditions that do not degrade the structure oraffect detection characteristics of the released molecular tag, E.Whenever a cleaving probe is used in a homogeneous assay format,cleavable linkage, L, is cleaved by a cleavage agent generated by thecleaving probe that acts over a short distance so that only cleavablelinkages within an effective proximity of the proximity probe arecleaved. Typically, such an agent must be activated by making a physicalor chemical change to the reaction mixture so that the agent produces ashort lived active species that diffuses to a cleavable linkage toaffect cleavage.

In a non-homogeneous format, because specifically-bound bindingcompounds are separated from unbound binding compounds, a widerselection of cleavable linkages and cleavage agents are available foruse. Cleavable linkages may not only include linkages that are labile toreaction with a locally acting reactive species, such as hydrogenperoxide, singlet oxygen or the like, but also linkages that are labileto agents that operate throughout a reaction mixture, such asbase-labile linkages, photocleavable linkages, linkages cleavable byreduction, linkages cleaved by oxidation, acid-labile linkages andpeptide linkages cleavable by specific proteases. References describingmany such linkages include Greene and Wuts, 1991, Protective Groups inOrganic Synthesis, Second Edition, John Wiley & Sons, New York;Hermanson, 1996, Bioconjugate Techniques, Academic Press, New York; andU.S. Pat. No. 5,565,324, each of which is incorporated by reference intheir entireties herein.

Molecular tag, E, in the present invention may comprise an electrophorictag as described in the following references when separation ofpluralities of molecular tags are carried out by gas chromatography ormass spectrometry: See, e.g., Zhang et al., 2002, Bioconjugate Chem.13:1002-1012; Giese, 1983, Anal. Chem. 2:165-168; and U.S. Pat. Nos.4,650,750; 5,360,819; 5,516,931; and 5,602,273, each of which is herebyincorporated by reference in its entirety.

Molecular tag, E, is preferably a water-soluble organic compound that isstable with respect to the active species, especially singlet oxygen,and that includes a detection or reporter group. Otherwise, E may varywidely in size and structure. In one embodiment, E has a molecularweight in the range of from about 50 to about 2500 daltons, morepreferably, from about 50 to about 1500 daltons. E may comprise adetection group for generating an electrochemical, fluorescent orchromogenic signal. In embodiments employing detection by mass, E maynot have a separate moiety for detection purposes. Preferably, thedetection group generates a fluorescent signal.

Molecular tags within a plurality are selected so that each has a uniqueseparation characteristic and/or a unique optical property with respectto the other members of the same plurality. In one embodiment, thechromatographic or electrophoretic separation characteristic isretention time under a set of standard separation conditionsconventional in the art, e.g., voltage, column pressure, column type,mobile phase or electrophoretic separation medium. In anotherembodiment, the optical property is a fluorescence property, such asemission spectrum, fluorescence lifetime or fluorescence intensity at agiven wavelength or band of wavelengths. Preferably, the fluorescenceproperty is fluorescence intensity. One or two or more of the moleculartags of a plurality may have identical migration or retention times, butthey will have unique fluorescent properties, e.g., spectrallyresolvable emission spectra, so that all the members of the pluralityare distinguishable by the combination of molecular separation andfluorescence measurement.

Preferably, released molecular tags are detected by electrophoreticseparation and the fluorescence of a detection group. In suchembodiments, molecular tags having substantially identical fluorescenceproperties have different electrophoretic mobilities so that distinctpeaks in an electropherogram are formed under separation conditions.Preferably, pluralities of molecular tags of the invention are separatedby a conventional capillary electrophoresis apparatus, either in thepresence or absence of a conventional sieving matrix. During or afterelectrophoretic separation, the molecular tags are detected oridentified by recording fluorescence signals and migration times (ormigration distances) of the separated compounds or by constructing achart of relative fluorescent and order of migration of the moleculartags (e.g., as an electropherogram). Preferably, the presence, absenceand/or amounts of molecular tags are measured by using one or morestandards.

A cleavage-inducing moiety, or cleaving agent, is a group that producesan active species that is capable of cleaving a cleavable linkage,preferably by oxidation. Preferably, the active species is a chemicalspecies that exhibits short-lived activity so that its cleavage-inducingeffects are only in the proximity of the site of its generation. Eitherthe active species is inherently short lived, so that it will not createsignificant background beyond the proximity of its creation, or ascavenger is employed that efficiently scavenges the active species, sothat it is not available to react with cleavable linkages beyond a shortdistance from the site of its generation. Illustrative active speciesinclude singlet oxygen, hydrogen peroxide, NADH and hydroxyl radicals,phenoxy radical, superoxide and the like. Illustrative quenchers foractive species that cause oxidation include polyenes, carotenoids,vitamin E, vitamin C, amino acid-pyrrole N-conjugates of tyrosine,histidine and glutathione. See, e.g., Beutner et al., 2000, Meth.Enzymol. 319:226-241.

One consideration in designing assays employing a cleavage-inducingmoiety and a cleavable linkage is that they not be so far removed fromone another when bound to a receptor complex that the active speciesgenerated by the cleavage-inducing moiety cannot efficiently cleave thecleavable linkage. In one embodiment, cleavable linkages preferably arewithin about 1000 nm and preferably within about 20-200 nm of a boundcleavage-inducing moiety. More preferably, for photosensitizercleavage-inducing moieties generating singlet oxygen, cleavable linkagesare within about 20-100 nm of a photosensitizer in a receptor complex.One of ordinary skill in the art will recognize that the effectiveproximity of a particular sensitizer may depend on the details of aparticular assay design and may be determined or modified by routineexperimentation.

A sensitizer is a compound that can be induced to generate a reactiveintermediate, or species, usually singlet oxygen. Preferably, asensitizer used in accordance with the invention is a photosensitizer.Other sensitizers included within the scope of the invention arecompounds that on excitation by heat, light, ionizing radiation orchemical activation will release a molecule of singlet oxygen. The bestknown members of this class of compounds include the endoperoxides suchas 1,4-biscarboxyethyl-1,4-naphthalene endoperoxide,9,10-diphenylanthracene-9,10-endoperoxide and 5,6,11,12-tetraphenylnaphthalene 5,12-endoperoxide. Heating or direct absorption of light bythese compounds releases singlet oxygen. Further sensitizers aredisclosed by Di Mascio et al., 1994, FEBS Lett. 355:287-289; Kanofsky,1983, J, Biol. Chem. 258:5991-5993; Pierlot et al., 2000, Meth. Enzymol.319:3-20.

Photosensitizers may be attached directly or indirectly, via covalent ornon-covalent linkages, to the antibodies. Guidance for constructing suchcompositions is available in the literature, e.g., in the fields ofphotodynamic therapy, immunodiagnostics and the like. Exemplary guidancemay be found in Ullman et al., 1994, Proc. Natl. Acad. Sci. USA 91,5426-5430; Strong et al., 1994, Ann. New York Acad. Sci. 745: 297-320;Yarmush et al., 1993, Crit. Rev. Therapeutic Drug Carrier Syst. 10:197-252; and U.S. Pat. Nos. 5,709,994, 5,340,716, 6,251,581, and5,516,636.

A large variety of light sources are available to photo-activatephotosensitizers to generate singlet oxygen. Both polychromatic andmonochromatic sources may be used as long as the source is sufficientlyintense to produce enough singlet oxygen in a practical time duration.The length of the irradiation depends on the nature of thephotosensitizer, the nature of the cleavable linkage, the power of thesource of irradiation and its distance from the sample. In general, theperiod for irradiation may be less than about a second to as long asabout 3 hours and is usually in the range of 15 minutes to 2 hours.Exemplary light sources include lasers such as, e.g., helium-neonlasers, argon lasers, YAG lasers, He/Cd lasers and ruby lasers;photodiodes; mercury, sodium and xenon vapor lamps and incandescentlamps such as, e.g., tungsten and tungsten/halogen and flashlamps. Anexemplary photoactivation device suitable for use in the methods of theinvention is disclosed International Patent Publication No. WO03/051669, which is incorporated by reference herein, including anydrawings. In such embodiments, the photoactivation device is an array oflight emitting diodes (LEDs) mounted in housing that permits thesimultaneous illumination of all the wells in a 96-well plate.

Examples of photosensitizers that may be utilized in the presentinvention are those that have the above properties and those disclosedby U.S. Pat. Nos. 5,536,834, 5,763,602, 5,565,552, 5,709,994, 5,340,716,5,516,636, 6,251,581 and 6,001,673; published European PatentApplication No. 0484027; Martin et al., 1990, Methods Enzymol.186:635-645; and Yarmush et al., 1993, Crit. Rev. Therapeutic DrugCarrier Syst. 10:197-252, each of which is incorporated by referenceherein in their entireties, including any drawings. As with sensitizers,in certain embodiments, a photosensitizer may be associated with a solidphase support by being covalently or non-covalently attached to thesurface of the support or incorporated into the body of the support. Ingeneral, the photosensitizer is associated with the support in an amountnecessary to achieve the necessary amount of singlet oxygen. Generally,the amount of photosensitizer is determined empirically according toroutine methods.

Following cleavage, the sample can then be analyzed to determine theidentity of molecular tags that have been released. Where an assayemploying a plurality of binding compounds is employed, separation ofthe molecular tags will generally precede their detection. The methodsfor both separation and detection are determined in the process ofdesigning the molecular tags for the assay. A preferred mode ofseparation employs electrophoresis, in which the various tags areseparated based on known differences in their electrophoreticmobilities.

In a second aspect, the invention is drawn to a method for determiningwhether a subject with a cancer is likely to respond to treatment with atargeted therapy, for predicting a time course of disease and/or forpredicting the probability of a significant event in the time course ofthe subject's cancer, comprising measuring in a biological sample fromthe subject's cancer an amount of Her-3, wherein the method is dependenton the level of Her-3. In certain embodiments, if the level of Her-3 ishigh, the patient is less likely or unlikely to respond to the targetedtherapy. In certain embodiments, if the level of Her-3 is low, thepatient is more likely to respond to the targeted therapy. In certainembodiments, as described in more detail herein, the therapy is a Heracting agent. In further embodiments, the therapy is at least one of aHer-2 acting agent or a Her-3-targeted agent.

In a certain embodiments, the breast cancer is early stage (i.e.,adjuvant) breast cancer or metastatic breast cancer. In certainembodiments, the level of Her-2 expression in the breast cancer is high.In certain embodiments, high Her-2 expression is a log 10H2T≧ about1.14-1.25. In certain embodiments, the high Her-2 expression comprisesexpression that is very high and/or moderately high. In certainembodiments, the very high Her-2 expression is a log 10H2T≧ about1.84-2.21. Or, other ranges may be used depending upon the patientcohort.

In a preferred embodiment, a time course is measured by determining thetime between significant events in the course of a patient's disease,wherein the measurement is predictive of whether a patient has a longtime course. In a preferred embodiment, the significant event is theprogression from primary diagnosis to death. In a preferred embodiment,the significant event is the progression from primary diagnosis tometastatic disease. In a preferred embodiment, the significant event isthe progression from primary diagnosis to relapse. In a preferredembodiment, the significant event is the progression from surgery todeath. In a preferred embodiment, the significant event is theprogression from surgery to relapse. In a preferred embodiment, thesignificant event is from surgery to metastases. In a preferredembodiment, the significant event is the progression from metastaticdisease to death. In a preferred embodiment, the significant event isthe progression from metastatic disease to relapse. In a preferredembodiment, the significant event is the progression from relapse todeath. In a preferred embodiment, the time course is measured withrespect to overall survival rate, time to progression and/or using theRECIST or other response criteria.

The Her-3 VERATAG® assay was used to examine the Her-3 levels in acohort of patients from the International Serum Her-3/neu Study Grouptrial. These patients (n=105) were selected primarily by IHC for Her-2positivity performed at a central location by a single pathologist andall received trastuzumab. Only patients with Her-2 over-expressingtumors (>10% of tumor cells IHC 3+ as determined by HercepTest®) and/orErbB2-amplified (with positive FISH testing mandatory on all IHC 2+cases) metastatic breast cancer were included in this study (see Example14 for additional details). The Her3 VERATAG® assay was performed onthese patient samples and out of 105 samples on which the assay wasperformed, 85 had measurable Her-3 levels above the limit of detection(8 had no detectable tumor, 1 sample had a fluorescein failure, and 8samples were below the limit of detection and considered low/negative inthe assay). The results are shown in FIG. 13.

Her-3 levels and in particular Her-3 levels in Her-2 positive tumorshave been implicated in having prognostic value with respect to the timecourse of disease progression and overall survival as well as responseto therapy and particularly with respect to escape fromEGFR-family-targeted therapeutics. See Sergina et al. (2007) Nature445:437-441, Osipo et al. (2007) Int J Oncol. 30:509-520, De Alava etal. (2007) Clinical Oncol. 25:2656-2663, Ma and Bose (2008) E-Updates inHER1 and HER2Targeting in Breast Cancer, Volume 2, Tovey et al. (2006)J. Pathol. 210:358-362, Menendez and Lupu (2007) Breast Cancer Res.9:111, Fuchs et al. (2006) Anticancer Res. 26:4397-4402, Lee-Hoeflich etal. (2008) Cancer Res. 68:5878-5886.

Her-3 expression has been examined in many cancers, including tumors ofpatients treated with therapeutics targeted to EGFR family members(e.g., trastuzumab, pertuzumab, lapitinib, cetuximab, gefitinib anderlotinib) as well as chemotherapeutics. In comparing Her-family memberlevels with clinical outcome, data suggest specifically that Her-3expression and/or the relative amounts of Her-2 and Her-3 may be of useas a prognostic and predictive diagnostic biomarker in ovarian cancer(see Amier et al. (2008) J. Clin. Oncol. 26:abstract 5552, Amier et al.(2008) Meeting: 2008 Molecular Markers, abstract 25, and Xu et al.(1999) Clin. Cancer Res. 5:3652-3660); and non-small-cell lung cancer(Cappuzzo et al. (2005) Brit. J. Cancer 93:1334-1340). In a preferredembodiment, the method further comprises determining whether the levelof Her-3 is high or low by dividing a sample group of Her-2-positivepatients into at least two subgroups comprising one subgroup with a highamount of Her-3 and at least one other subgroup with a low amount ofHer-3, wherein if the Her-3 is low, then the patient is likely torespond to Her-2-targeted therapy, the time course of the disease islikely to be long and the patient is not likely to have a significantevent. In a preferred embodiment, the subject's cancer is breast cancer,colorectal cancer, ovarian cancer, bladder cancer, prostate cancer,non-small cell lung cancer, melanoma, pharyngeal cancer, pancreaticcancer, esophageal cancer, glioma, bile duct carcinoma, biliary tractcarcinoma, cholangiocarcinoma, gastric cancer, endometrial cancer, gallbladder cancer, squamous cell carcinoma or basal cell carcinoma. In apreferred embodiment, the subject's cancer is breast cancer, melanoma,colorectal cancer or ovarian cancer. In a preferred embodiment, thesubject's cancer is a Her-2 positive breast cancer. In a preferredembodiment, the breast cancer is early stage (i.e., adjuvant) breastcancer or metastatic breast cancer.

In a preferred embodiment, the targeted therapy is at least one Herfamily-targeted agent. In a preferred embodiment, the Herfamily-targeted agent is a multi- or single-targeted agent. In apreferred embodiment, the multi-targeted agent is a dual kinaseinhibitor or a bispecific antibody. In a preferred embodiment, the Herfamily targeted agent is trastuzumab, lapatinib or pertuzumab. In apreferred embodiment, the at least one Her family-targeted agent is atleast two agents, wherein the at least two agents are one or moreHer-2-targeted monoclonal antibodies and/or EGFR-targeted monoclonalantibodies and/or an EGFR and Her-2 dual kinase inhibitor. In apreferred embodiment, the monoclonal antibody is trastuzumab. In apreferred embodiment the EGFR-targeted monoclonal antibody is cetuximabor panitumumab. In a preferred embodiment, the EGFR-targeted monoclonalantibody is zalutumumab, nimotuzumab, and matuzumab. In a preferredembodiment, the dual kinase inhibitor is lapatinib, erlotinib orgefitinib. In a preferred embodiment, the targeted therapy is a Her-3 orHer-3 signaling pathway acting agent. In a preferred embodiment, theHer-3 or Her-3 signaling pathway targeted agent is a Her-3 monoclonalantibody, a Her-3 dimerization inhibitor, a Her-3 phosphorylationinhibitor and/or an inhibitor of a Her-3 signaling pathway memberselected from the group consisting of PI3K, Akt, mTOR, ERK1/2, or PYK2.In a preferred embodiment, likeliness to respond, likeliness to have along time course and/or likeliness to have a significant event ismeasured as an overall survival rate, as time to progression, asdisease-free survival, as progression-free survival, and/or as objectivetumor response using the RECIST criteria. Signal transduction refers toany process by which cells convert one kind of signal into another.Typically, this involves some type of signal on the cell surface (forexample, the binding of a ligand to a cell surface receptor), followedby a cascade of biochemical reactions inside the cell, which are carriedout by enzymes, resulting in a signal transduction pathway or signalingpathway, effecting a multitude of cellular functions. In the case of theEGFR family of tyrosine kinase receptors, each of the four receptors inthe family has an extracellular domain, comprising both a dimerizationdomain and a ligand-binding domain, as well as a trans-membrane domainand an intracellular domain with tyrosine kinase activity (see Burgesset al. (2003) Mol Cell. 12:541-542). In Her-3, the kinase domain is notfunctional but through dimerization with other family members, Her-3 canexert significant signaling pathway effects. Evidence suggests thatcooperation of multiple ErbB receptors and ligands is required forinitiating cell transformation. When activated, this family of receptorssustains a complex network of signaling pathways. All EGFR familymembers have been found to be expressed and/or altered in a variety ofcancers and may play a significant role in tumor development, includingproliferation, apoptosis, and metastasis (see Burgess (2008) GrowthFactors 26:263-274 and Normanno et al. (2006) Gene 366:2-16). Intenseinterest in targeting the EGFR family members (see Bianco et al. (2007)Int. J. Biochem. Cell. Biol. 39:1416-1431), particularly EGFR and Her-2,has resulted in several approved targeted therapeutics. Based onpromising preclinical data in both in vitro and in vivo test models, theresults in clinical trials have been somewhat disappointing, resultingin increased interest in other family members such as Her-3, as well asdownstream signaling pathway members.

Her-3 signaling has been linked to cancer and, in particular,Her-2/Her-3 dimer formation may be crucial for increased aggression intumors that over-express Her-2, leading to interest in targetedtherapeutics that inhibit dimer formation as well as downstream pathwaysactivated by Her-3. Her-3 is particularly adept at signaling because ithas 6 binding sites for phosphoinositide 3′-kinases (PI3K) which inturn, activate protein kinase B (also called AKT). The “PI3K/AKT”signaling pathway has been shown to be required for an extremely diversearray of cellular activities—most notably cellular proliferation andsurvival—fueling the interest in targeting both Her-3 as well asdownstream signaling pathway members to create novel targetedtherapeutics or to potentiate the therapeutic value of currentEGFR-family targeted therapeutics. (For review, see Stern (2008) JMammary Gland Biol Neoplasia 13:215-223, Sithanandam and Anderson (2008)Cancer Gene Ther. 15:413-448 and Arkin and Moasser (2008) Curr. Opin.Investig. Drugs 9:1264-1276).

In a preferred embodiment, whether the cancer is Her-2 positive isdetermined by IHC, FISH, CISH, quantitative mRNA, hybridization array orVERATAG®. In a preferred embodiment, determining the level of Her-3 isperformed using IHC, FISH, CISH, quantitative mRNA, hybridization arrayor VERATAG®. In a preferred embodiment, the method further comprisesdetermining whether an amount of Her-3 protein is low by comparing theamount of Her-3 in the subject's cancer to a pre-determined cutoff. In apreferred embodiment, the method further comprises determining the levelof Her-3 by dividing a sample group of Her-2-positive patients into atleast two subgroups comprising one subgroup with a high amount of Her-3and at least one other subgroup with a low amount of Her-3, wherein ifthe Her-3 is high, then the patient is unlikely to respond toHer-2-targeted therapy, the time course of the disease is likely to beshort and/or the patient is likely to have a significant event.

In a preferred embodiment, the method further comprises determining thata subject is afflicted with a Her-2 positive cancer that is unlikely torespond to treatment according to a method of the invention, thenadvising a medical professional of the treatment option of administeringto the subject an effective amount of a different therapeutic agent.

In a third aspect, the invention is drawn to a purified antibody thatbinds to Her-3. In a preferred embodiment, the antibody is a polyclonalantibody or a monoclonal antibody. In a preferred embodiment, theantibody is a monoclonal antibody. In a preferred embodiment, theantibody is raised against one of the peptides having SEQ ID NOs:1-8, asset forth in Example 2 and shown in FIG. 2A. In a preferred embodiment,the antibody is a monoclonal antibody comprising (a) a light chainvariable region comprising CDR1, CDR2 and CDR3 having sequences as setforth in SEQ ID NOs:13, 14 and 15, respectively, and (b) a heavy chainvariable region comprising CDR1, CDR2 and CDR3 having sequences as setforth in SEQ ID NOs:16, 17 and 18, respectively; and/or a monoclonalantibody comprising (a) a light chain variable region comprising CDR1,CDR2 and CDR3 having sequences as set forth in SEQ ID NOs:19, 20 and 21,respectively, and (b) a heavy chain variable region comprising CDR1,CDR2 and CDR3 having sequences as set forth in SEQ ID NOs:22, 23 and 24,respectively (Table 1B). In a preferred embodiment, the antibody is theantibody with the amino acid sequence having SEQ ID NOs:9 and 11 as setforth in Table 1A for the light and heavy chains, respectively, and/orSEQ ID NOs:10 and 12 as set forth in Table 1A for the light and heavychains, respectively.

In a preferred embodiment, the invention is drawn to the DNA encodingthe antibody. The DNA encoding the monoclonal antibodies is isolated andsequenced using techniques commonly known to those skilled in the art ofcloning. Once isolated, the DNA can be ligated into expression vectorsand transfected into appropriate host cells to obtain recombinantantibodies from cultured cells (see Plueckthun (1992) Immunological Rev.130: 151-188).

Those with skill in the art will appreciate that the amino acid sequenceof an antibody can be modified and that modifications may be desirableto enhance the properties of the antibody for therapeutic, analytical ordiagnostic use. Further it will be appreciated that one or more aminoacids in these antibodies may be changed by insertion, deletion orsubstitution without appreciably diminishing the binding characteristicsof the antibody. Exemplary amino acid changes would be substitutionsusing amino acids with similar molecular characteristics (i.e.,conservative substitutions, e.g., changing amino acids from within thefollowing subgroups of aromatic amino acids, acidic amino acids, basicamino acids or amino acids with amides or sulphurs). Othernon-conservative substitutions or insertions may be made withoutappreciably altering molecular integrity or binding characteristics.Further, some amino acid changes or collection of amino acid changeswill enhance properties of the antibody, including but not limited to,better binding affinity, greater stability, (e.g., resistance toproteases) selectivity and/or ease of production. Methods for changingamino acid sequences and/or selecting for molecules with betterproperties are known to those with skill in the art. Preferably, inintact antibodies, the degree of sequence identity after modification isat least 50% and more preferably, at least 75% and most preferably atleast 90-95%. Each of these antibodies is intended to be within thescope of the contemplated invention.

In a preferred embodiment, antibodies targeted to Her-3 may be used todevelop additional Her-3-targeted molecules. Modifications of theantibodies described herein may be desirable to improve qualitiesincluding, but not limited to, increasing effector function, decreasingimmunogenicity, increasing stability, improving pharmacologic propertiessuch as serum half-life and aiding in ease and yield of production. Eachof these targeted molecules is intended to be within the scope of thecontemplated invention.

In a preferred embodiment, humanized antibodies comprising the antigenbinding regions of the antibodies described herein (see Table 1A) in ahuman framework may be used for therapeutic applications. Severalmethods for humanizing antibodies have been reported (see Jones et al.(1986) Nature 321:522-525, Riechmann et al. (1988) Nature 332:323-327and Verhoeyen et al. (1988) Science 239:1534-1536). Typically, thenon-human sequences of the variable domain are screened computationallyagainst the entire repertoire of human light and heavy chain variabledomain sequences to find the human variable framework sequences closestto the rodent sequences (see Sims et al. (1993) J. Immunol.151:2296-2308, Chothia et al. (1987) J. Mol. Biol. 186:901-917).Alternatively, consensus frameworks can be used (see Carter et al.(1992) Proc. Natl. Acad. Sci. USA 89:4285-4289 and Presta et al. (1993)J. Immunol. 151:2623-2632). In a preferred embodiment, computer-aideddesign is used to select sequences that confer stability and retain orimprove binding characteristics. Each of these is intended to be withinthe scope of the contemplated invention.

In another embodiment, the antibody CDRs may be used to create targetedbinding molecules that bind the same epitope in Her-3 but are containedwithin a framework that is not a native antibody. For example, oneskilled in the art would appreciate that methods are available forcreating binding molecules in which the framework may be a portion of anantibody, for example, an scFv or F(ab′)2 (see WO 93/16185 and Carter etal. (1992) Bio/Technology 10:163-167, respectively), each of which isincorporated by reference herein. One skilled in the art may alsoappreciate that a completely unrelated protein (such as a bacterialbeta-lactamase) can properly display the binding domain(s) to form abinding compound. In this sense, related antibodies, as defined herein,are intended to be within the scope of the invention.

The antibody may act therapeutically through binding alone or throughother properties (e.g., enzymatic activity or toxic warheads). In oneembodiment, the targeted protein may be modified to exert a therapeuticeffect or a greater therapeutic effect via antigen-dependentcell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity(CDC). In another embodiment, toxins may be conjugated to the antibodyor targeted protein. Exemplary small molecule toxins include but are notlimited to maytansine, calicheamicin and CC-1065 (see, e.g., Carter andSenter (2008) Cancer J. 14:154-169). Additionally, radiolabels can belinked to antibodies to create targeted therapeutics. Biologic toxinsmay also be linked to targeted proteins and include, but not be limitedto, diphtheria toxin, Pseudomonas exotoxin, abrin and ricin (seeKreitman (2006) AAPS J. 18:E532-551)

In a further embodiment, the targeted antibodies (or fragments thereof)may be fused to enzymes for use in antibody-directed enzyme prodrugtherapy (ADEPT; see Bagashawe (1987) Br. J. Cancer 58:700-703 and Senteret al. (1988) Proc. Natl. Acad. Sci. USA 85:4842-4846). In anotherembodiment, the antibodies or targeted proteins may be fused tomolecules such as polyethylene glycol that enhance pharmacologicproperties (e.g., serum half-life) (see Harris and Chess (2003) Nat.Rev. Drug Discov. 2:214-221).

EXAMPLES

The present invention may be better understood by reference to thefollowing non-limiting examples.

Example 1 Generation of Stable Cell Lines Expressing Varying Levels ofHER3 Protein

A commercially available cDNA (Origene Technologies, Inc.) forfull-length HER3 (GenBank No. NM_(—)001982.2) was digested with therestriction enzymes Not I and Xba I and the resulting fragment wassubcloned into pcDNA.3 1 Zeocin selectable expression vector. Theresulting plasmid was transformed into bacteria and screened to verifythe correct insert. Positive clones were sequence verified, expanded inbacteria and the plasmid purified using the Qiagen Maxi-prep kit. Humanembryonic kidney cells (HEK-293) were purchased from the American TypeCulture Collection and maintained in DMEM supplemented with 10% FBS, 1×penicillin-streptomycin (100× is 10,000 U/mL penicillin-G and 10,000μg/ml streptomycin), and Glutamax® (GIBCO) at 37° C. in 5% CO₂. The dayprior to transfection, the cells were split to approximately 25-30%confluence and incubated overnight in media without pen-strep. The cellswere then transfected with Fugene® HD (Roche) according to themanufacturer's instructions. The next day the media was replaced withfresh complete media and the cells were incubated for 48 hours prior tothe addition of 400 mg/mL Zeocin® (Invitrogen) in complete media. Theconcentration of Zeocin was determined by performing a killing curveusing varying concentrations of Zeocin on wild-type 293 cells that donot contain the transfected plasmid. Approximately 16 Zeocin®-resistantclones were isolated using cloning rings and frozen in liquid nitrogen.A subset of the original clones could be expanded successfully andtested using an HER3-specific ELISA kit (R & D Systems, Inc), accordingto the manufacturers instructions and verified as over-expressing HER3(FIG. 1). One clone with high expression of HER3 as demonstrated byELISA (293-H3 clone 1) was selected as a control for use in theoptimized assay.

Example 2 Generation and Screening of Antibodies Against HER3

A HER3-specific monoclonal antibody (Ab-6) with epitope specificity tothe cytoplasmic terminus of HER3 was purchased from LabVision. TheVERATAG® reporter (Pro11) and streptavidin-conjugated methylene blue(“molecular scissors”) were synthesized and purified according toprotocol described previously (see, for example, above and U.S. Pat. No.7,105,308, which is incorporated by reference herein, including anydrawings). Antibody-VERATAG® and antibody-biotin conjugates, i.e.,Ab6-Pro11 and B9A11-biotin, were made using sulfo-NHS-LC-LC-biotin(Pierce) as linker according to manufacturer's protocol and conjugationproducts purified by HPLC (Agilent). A series of proprietary antibodieswere generated as follows. Mice were immunized with fixed 293 clone 13cells or a series of peptides representing different epitopes containedwithin the c-terminal region of the HER3 protein (FIG. 2A). During theimmunization period, each mouse received several immunizations over a 4week period. Hybridomas were produced and clones isolated using limitingdilution. Conditioned media from individual clones were profiled by aseries of assays, including CellSpot™ or ELISA screening, followed byscale-up and then further screening by immunohistochemistry (IHC) (FIG.2B), and then finally by VERATAG® Technology using either a chemicalrelease method (Methylene Blue) (FIG. 2C) or the dual-antibody lightrelease method (FIG. 2D). Several antibodies that performed well usingthese methods and are described in FIG. 2A. One in particular, B9A11,was the most robust and gave the best dynamic range and sensitivity andwas carried forward to develop the final format of the assay asdescribed below in Example 5.

Example 3 Generation of Blocks and FFPE Sections from a Panel of CellLines Expressing Varying Levels of HER3 by FACS and ELISA

Three cell lines with varying expression of HER3 protein, MDA-MB-453,MDA-MB-468 and SKOV-3, were purchased from American Type Cell CultureCollection. MDA-MB-453 and MDA-MB-468 cell lines were maintained at 37°C. and 5% CO₂ in Dulbecco's modified Eagle medium (DMEM), 10% FBS, 1×penicillin-streptomycin and 1× Glutamax®. SKOV3 cells were maintained at37° C. and 5% CO₂ in McCoy's 5a Media supplemented with 10% FBS, 1×penicillin-streptomycin and 1× Glutamax (GIBCO). 293-H3 clone 1 cellswere maintained at 37° C. and 5% CO₂ in DMEM supplemented with 10% FBS,1× penicillin-streptomycin and 1× Glutamax. Cells were grown to nearconfluence on at least ten 500 mm culture plates for each cell line.After removal of medium, the cells were washed once with cold 1×PBS and15 mL of 1× Pen-fix (Thermo Scientific) was added to each plate. Cellswere scraped and the cell solution fixed overnight (>16 hrs) at 4° C.Following the overnight fixation the cells were centrifuged at 3200×gfor 15 min. The cell pellet was transferred to a rubber O-ring, wrappedwith filter paper and placed in a processing cassette. An automaticTissue-Tek processor was used for processing. Briefly, the cell pelletwas exposed to increasing concentrations of alcohol, Clear-rite (xylenesubstitute) and paraffin. After processing, the pellet was embedded in ablock using a paraffin embedding station. All solvents used for cellpellet processing were obtained from Richard-Allen Scientific. Theproportion of the same lot of cells was tested for HER3 receptor numberusing flow cytometry by the following method. A whole cell lysate wasprepared from the preparation of cells for quantifying levels of HER3receptor by using a commercially available ELISA kit and followingmanufacturer's recommendations (Human ErbB3-DuoSet ELISA; R & Dsystems). Sections of 7 um in thickness were sliced with a microtome(LEICA) and placed on positively charged glass slides (VWR). Slides wereair-dried for 30 min and then baked in a heated oven set at 70° C. for1.5 hr. All sample slides were stored at 4° C. for future assays.Results of the ELISA, flow cytometry, IHC and VERATAG® comparison areshown in FIG. 3.

Example 4 Generation of Sections from Commercially Available BreastCancer FFPE Sections

FFPE breast cancer blocks were purchased from Asterand. Sections of 5 umin thickness were sliced with a microtome (LEICA) and placed onpositively charged glass slides (VWR). Sections were air-dried for 30min and then baked in an oven at 70° C. for 1.5 hr. All sample slideswere stored at 4° C. for future assays. Previously sectioned breasttumors from clinical material were also used for these studies. Examplesof H & E stained tumors on glass slides are shown in FIG. 4.

Example 5 Her-3 VERATAG® Assay in Formalin Fixed, Paraffin Embedded(FFPE) Cell Lines and Breast Tissue

FFPE samples were deparaffinized/rehydrated using a series of solvents.Briefly, slides were sequentially soaked in xylene (2×, 5 min), 100%ethanol (2×, 5 min), 70% ethanol (2×, 5 min) and deionized water (2×, 5min). Heat-induced epitope retrieval of the rehydrated samples wasperformed in a slide holder containing 250 mL of 1×DAKO (pH 9.0) (LabVision) using a pressure cooker (Biocare). After being cooled for 30 minat room temperature, the slides were rinsed once with deionized water. Ahydrophobic circle was drawn on slide using a hydrophobic pen (Zymed) toretain reagents on slides. The samples were then blocked for 1 hr withblocking buffer that contains 1% mouse serum, 1.5% BSA and a cocktail ofprotease and phosphatase inhibitors (Roche) in 1×PBS. After removal ofthe blocking buffer with aspiration, a mixture of VERATAG®-conjugated(Ab-6: LabVision, 1 ug/mL) and biotin-conjugated (B9A1 1; Monogramproprietary, 2 ug/mL) antibodies prepared in blocking buffer was addedand binding reactions were incubated overnight in a humidified chamberat 4° C. with shaking. The antibody mix was aspirated and samples werewashed with wash buffer containing 0.25% TritonX-100 in 1×PBS andstreptavidin-conjugated methylene blue at concentration of 2.5 ug/mL in1×PBS was added. After 1 hr incubation at room temperature, the excessstreptavidin-methylene blue reagent was aspirated and the samples werewashed in wash buffer once followed by 3 changes of deionized water.Illumination buffer containing 3 μM fluorescein and two CE internalmarkers (MF and ML) in 0.01×PBS was added on sample sections. The boundVERATAG® was released at ˜4° C. by photo-activated cleavage using anin-house LED array illuminator equipped with an electronic chiller block(Torrey Pine Scientific). After illumination, VERATAG® intermediates arereduced to a quantifiable form by the addition of sodium borohydride.The CE sample containing the released VERATAG® reporters was collectedfrom above the tissue section on the slides and the released VERATAG®reporters in the CE samples were separated and detected on ABI3130 CEinstrument (22-cm capillary array; Applied Biosystems) under CEinjection condition of 6 kV and 50 sec at 30° C. The general workflow ofthe H3T assay in the clinical lab is illustrated in FIG. 5.

Example 6 CE Peak Analysis, Tumor Area Normalization and BatchNormalization

The identification and quantification of VERATAG® was carried out usingVERATAG® Informer software (see, for example, United States publicationnumber 2007-0203408-A1). To analyze the VERATAG® signals in a raw CEelectropherogram, two CE internal markers, MF (first marker) and ML(last marker), were used to identify the VERATAG® peaks according totheir electrophoretic mobility or migration time, t, relative to the twomarkers, i.e., [t(VERATAG®)-t(MF)]/[t(ML)-t(MF)]. The identifiedVERATAG® peaks were then quantified by peak area calculation for eachVERATAG®. To correct for variability in VERATAG® recovery from thetissue section, and the run variability in CE injection efficiencyand/or detection sensitivity across capillary array, fluorescein (3 pM)was included in the illumination buffer, and co-electrophoresed as aninternal reference control in each sample run. The area of each VERATAG®peak is then reported as RFU or RPA by area normalization of theVERATAG® peak (VERATAG® peak area) to the internal fluorescein peak(fluorescein peak area/3 μM. This is quantified as RPA*IB vol/TA forvariable tumor samples (=Relative peak area multiplied by theillumination buffer volume (TB) loaded onto sample section; divided bythe tumor area in mm² (RPA*IB vol/TA=pmole/L*L/mm²=pmole/mm²).Specifically, the CE fluorescence signal intensity of a VERATAG®reporter, or the peak area (PA_(VeraTag®)), is given in relativefluorescent units (signal height) integrated across time (RFU-S/S). Therelative peak area(RPA_(VeraTag® is measured by normalizing the VERATAG® peak area (PA)_(VeraTag®)) with respect to the internal fluorescein standard of knownconcentration (PA_(F)), and is therefore proportional to the initialconcentration of the analyte being measured. As the VERATAG® assaysignal is a quantitative readout that scales with tumor content,accurate comparison of VERATAG® assay signals across clinical samplesrequires adjustment for differences in this parameter. Therefore, tumorcontent is measured on the sample VERATAG® assay signal data collection,and the tumor content is used to normalize the VERATAG® assay result. Itshould be noted that, if the tumor sample is either very small or verylarge the reaction volumes (i.e., volume of antibody,streptavidin-conjugated methylene blue and illumination buffer reagents)are adjusted, and this adjustment is reflected in the tumor contentnormalization. After adjusting VERATAG® peak areas for migration time,fluorescein, illumination buffer and tumor area, controls and samplesare normalized by multiplying their adjusted peak area with therespective calculated Batch Normalization Factor (BNF). Each adjustedRPA value is multiplied by the respective BNF to obtain a normalized RPAvalue. Because this normalized RPA value is unitless by definition, itsvalue is referred to in VERATAG® units. All reportable (not failed, andnot saturated) normalized values for a given sample are averaged todetermine a final value for that sample.

Various quality control checks have been developed to ensure data is ofhighest quality.

-   -   1. Fluorescein out of range. A sample trace with fluorescein out        of range is failed.

The fluorescein range is calculated by using the median of the adjustedfluorescein value +−XX %. The XX % value, typically 20-40%, isadjustable and is associated with individual templates. Sample peaks arefailed if the absolute height of the peak is >7000 units, referred to asa saturated peak.

-   -   2. RPA must be greater than or equal to 0.03.    -   3. If an undiluted converted peak sample is failed due to RPA        <0.03, then all of the corresponding converted peak diluted        samples are also failed.    -   4. At least 2 converted peak controls must have called values        for converted peak batch normalization to proceed.    -   5. A sample or control with poor trace quality is failed.    -   6. If illumination buffer-only controls have contamination,        samples may be failed if trace quality may be affected, e.g., if        the contaminating peak overlaps with the released peak    -   7. A batch with poor batch normalization is failed.    -   8. A batch with an abnormally high batch normalization factor is        failed.

For each sample, following the VERATAG® assay, an H&E (Hematoxylin andEosin) staining and evaluation is performed to assure presence of tumorcells and to enable an estimation of tumor area. These slides aredeparaffinized, hydrated, stained, and then dehydrated and mounted usingstandard procedures before microscopic examination.

Example 7 Optimization of Antibody Concentration to Increase DynamicRange

Optimal concentrations of the Ab-6 and the B9A1 1 antibody weredetermined by varying the final concentrations in the VERATAG® HER3total assay (as described above) on a cell line panel spanning theentire dynamic range of the assay (293H3-clone 1, MDA-MB-453,MDA-MB-468, and SKOV3). These results were then compared this withexpected fold changes based on HER3 flow cytometry and ELISA resultsfrom the same cell line FFPE block preparation. An optimal concentrationof 2 mg/mL B9A11-biotin and 1 mg/mL of Ab-6 Pro-11 was selected forperformance based on the accurate detection of HER3 as compared with theexpected fold changes as compared to ELISA and flow cytometry in thissame set of cell lines as shown in FIG. 6 where a 2:1 ratio of B9A11 toAb-6 is circled.

Example 8 Accuracy of HER3 Total VERATAG® Assay

One batch of the HER3 total VERATAG® assay was performed using threesuccessful replicates from four well-characterized cell lines(293H3-clone 1, MDA-MB-453, MDA-MB468, MDA-MB-231 and SKOV3) and thencompared for accuracy of VERATAG® measurement with in-house generatedflow cytometry and ELISA data. 100% of the results matched the in-housedata from flow cytometry and ELISA in that 293H3-clone1>MDA-MB-453>MDA-MB468>SKOV3. No overlap was observed between signallevels for any of the four cell line samples, i.e., each cell lineseparated completely. Internal datasets on HER3 Total levels weregenerated by both ELISA and flow cytometry. Results for the fouraccuracy cell lines are presented in FIG. 7. Results from HER3 flowcytometry were very similar to results from HER3 ELISA in that the samerank order preservation was demonstrated by using these cross-validatingtechnologies.

Example 9 Sensitivity of HER3 Total VERATAG® Assay

The sensitivity of the HER3 total VERATAG® assay was determined bycomparing one batch containing 8 replicates of the low HER3 expressioncontrol cell line, MDA-MB-468 with 8 replicates of the low/negative HER3expression control cell line, SKOV3. The values for each replicate ofMDA-MB-468 were compared with SKOV3 replicates by pairwise comparisonsto determine sensitivity. 100% (64/64) of the pairwise comparisonsbetween MDA-MB-468 and SKOV3 resulted in MDA-MB-468>SKOV3 (FIG. 8).

Example 10 Reproducibility of HER3 Total VERATAG® Assay

Inter-assay reproducibility was determined by performing 8 separatebatches of the HER3 total VERATAG® assay as described above on 4 wellcharacterized cell lines (293H3-clone 1, MDA-MB-453, MDA-MB-468 andSKOV3) using different instrumentation (CE, illuminators), severaloperators, and performing the assay over a 4 week period. Followingbatch normalization procedures, the data was compared across the 8batches to determine reproducibility. The reproducibility across thedynamic range was between 8-15% (see FIG. 9).

Example 11 Precision of HER3 Total VERATAG® Assay

Intra-assay reproducibility was determined by performing 1 batch/cellline of the HER3 total VERATAG® assay and comparing the performance of15 replicates of each of the 3 control cell lines (293H3-clone 1,MDA-MB-453, MDA-MB-468). Pairwise comparisons were made of the 15replicates in each batch to determine precision of the assay. 95% of the293H3-clone 1 data was within 1.2 fold, 100% of the MDA-MB-453 data waswithin 1.23-fold, and 95% of the MDA-MB-468 data is within 1.37-fold.Results are shown in FIG. 10.

Example 12 Linearity of the HER3 total VERATAG® Assay

The linearity of the HER3VERATAG® result was determined by takingwell-characterized cell line controls 293H3-clone 1, MDA-MB-453,MDA-MB-468) and performing successive “cut-down” experiments to createFFPE sections with the following dimensions 1, ½, ¼, 1/16. These“cut-down” sections were then run in the H3T VERATAG® assay and thefinal section area normalized data was compared in a pairwise manner tounderstand the linearity of the assay with respect to section size (FIG.11). MDA-MB-453 is linear down to approximately 1/16 of the originalsection size, while MDA-MB-468 is linear down to approximately should be½ of the original section size. Results are shown in FIG. 11.

Example 13 Specificity of the HER3 Total VERATAG® Assay

Patient-derived tumor samples and cell line controls were run using theVERATAG® HER3Total Assay and using isotype control antibodies. For theHER3Ab-6-Pro11, the matched isotype control was IgG1-Pro11. For theHER3B9A11-biotin, the matched isotype control was also IgG1-biotin.Signal from these reactions is not antigen-specific, and would representnon-specific background. Samples were run in each of the following threeformats and run within the same batch side by side:

Format 1: HER3Ab6-Pro11/B9A11-biotin (normal format)Format 2: HER2Ab6-Pro11/IgG1-biotinFormat 3: IgG1-Pro11/B9A11-biotin

Sample results from each IgG1 format (Format 2 and Format 3) werecompared to the negative control, SKOV3, present in each batch(comparison parameter A). Samples results were also compared to therespective actual HER3Total signals (Format 1). Results are shown inFIG. 12.

Example 14 Measurement of Clinical Breast Tumors and Dynamic Range

The study population comprised patients (n=105) that were prospectivelyobserved during trastuzumab-based therapy at a single institutionbetween 1999 and 2006 (the International Serum Her-2/neu Study Grouptrial). Only outpatients (ECOG PS 0-2, age >18 years, estimated lifeexpectancy >12 weeks) with HER-2/neu-overexpressing (>10% of tumor cellsIHC 3+ as determined by the HercepTest®; DAKO Diagnostics, Austria)and/or ERBB2-amplified (with positive FISH testing mandatory in all IHC2+ cases) MBC were included. In addition, patients were required to betrastuzumab-naïve and have bi-dimensionally measurable diseaseprogressing within 4 weeks before initiation of treatment (excludingpreviously irradiated lesions). Samples from this study group trial wererun in the H3T VERATAG® assay in eight separate batches. For each batch,CE peak analyses were performed, as well as tumor area analyses and thebatch was normalized accordingly. Out of the 105 patient samples run inthe assay, 85 samples had measurable H3T levels above the limit ofdetection, 8 patient samples had no detectable tumor, 1 sampledemonstrated a fluorescein failure excluding the data, and finally therewere 8 patient samples that were below the limit of detection of theassay and considered low/negative in H3T expression. The results areshown in FIG. 13.

Example 15 Determination of Optimal Cutoff for Trastuzumab Response

Using positional scanning analysis an optimal cut-off was determinedwhereby patients above statistically significant cut-off had anunfavorable time to progression (TTP) compared to patients that werebelow this cut-off (FIG. 14). The cut-off was ˜ the median of theresults of the population tested (0.158, HR=2.3, p=0.0004. TTP wasdefined as the time from the initiation of trastuzumab-containingtreatment to progression (SWOG) or censor, and OS was defined as thetime from initiation of trastuzumab-containing treatment to death orcensor. When looking at overall survival in this population of patientsno significant cut-off could be determined, however there was a trend inOS using the 0.158 cut-off (HR=1.7; p=0.059).

Example 16 Kaplan Meier (KM) Analysis for TTP

A previously reported H2T cutoff (log H2T≧1.14) was used to sub-dividethe patients into HER2− normal (N=26, median TTP=4.1 mos) andHER2-overexpressing (N=55, median TTP=11.1 mos, HR=0.43, p=0.0002)groups. In the HER2-overexpressing group, levels of H3T expression abovean optimal cutoff (H3T>0.158; FIG. 14), as defined by a positionalscanning predicted shorter median time to progression (N=25, medianTTP=6.1 mos) compared with H3T expression below the cutoff (N=30, medianTTP=13.1 mos, HR=2.7, p=0.0002). Univariate Cox proportional hazardsanalyses examining the HER2-overexpressing sub-group identified H3T(above vs below a particular cutoff) as the most significant predictorof TTP (HR=2.98, p=0.0004). These results are shown in FIG. 15.

Example 17 HER3 Total Expression by VERATAG® in Other Malignancies

H3T VERATAG® assay was performed on a number of different malignanciesother than breast cancer, including colon, ovarian, synovial tumors(FIG. 16). Similar dynamic ranges were observed for all of these cancerswith the following rank order of range: Ovarian>SynovialCarcinoma>/=Colon. The dynamic range in these tumors ranges from 0.5-1.5logs depending on the cancer.

Example 18 Measurement of HER3 in Conjunction with P95 and Correlationwith Trastuzumab Response

A previously reported P95 cut-off (U.S. patent application Ser. No.12/629,037) was used to further stratify the HER-2 over-expressingpatients described above after initial subdivision based on their levelof HER3 to produce 4 patient subgroups as shown in FIG. 17. Patientswith a low level of P95 and HER3, defined as below an optimal cut-offpredicted by positional scanning, had a longer median time toprogression (TTP=15.0 mos) than any of the other subgroups (logrank testfor trend p<0.0001). Patients with a high level of P95 and HER3 had theshortest median time-to-progression (TTP=3.2 mos). The results are shownin FIG. 17

Example 19 Measurement of HER2, HER3 and P95 and Correlation withTrastuzumab Response

The patient population in Example 17 was subdivided by their level ofHER2, P95 and HER3 as shown in FIG. 18. Patients who are HER2-high,P95-low, and HER3-low, as defined by a positional scanning methodology,had a longer time to progression than patients who have a high level ofHER2 and a high level of HER3 or P95 or both with the latter showing asimilar time to progression as patients who have a low HER2 value asdetermined by the H2T VERATAG® assay. Within the subgroup with normalHER2 expression levels (H2Tlo) FISH-positive and FISH-negative groupsexperienced a similar time to progress.

All publications and other materials described herein are used toilluminate the invention or provide additional details respecting thepractice and are incorporated by reference in their entirety.

What is claimed is:
 1. A method of measuring and/or quantifying the presence and/or amount of Her-3 or Her-3 in a complex in a sample from a subject, the method comprising providing a sample and determining the presence and/or quantity of Her-3 and/or Her-3 in a complex in the sample using a Her-3 antibody that specifically binds to a polypeptide sequence set forth in one of SEQ ID NOs:1-8.
 2. The method of claim 1, wherein the Her-3 antibody specifically binds to a polypeptide sequence set forth in SEQ ID NO:6.
 3. The method of claim 1, wherein the Her-3 antibody is at least one of a monoclonal antibody comprising (a) a light chain variable region comprising CDR1, CDR2 and CDR3 having sequences as set forth in SEQ ID NOs:13, 14 and 15, respectively, and (b) a heavy chain variable region comprising CDR1, CDR2 and CDR3 having sequences as set forth in SEQ ID NOs:16, 17 and 18, respectively; or a monoclonal antibody comprising (a) a light chain variable region comprising CDR1, CDR2 and CDR3 having sequences as set forth in SEQ ID NOs:19, 20 and 21, respectively, and (b) a heavy chain variable region comprising CDR1, CDR2 and CDR3 having sequences as set forth in SEQ ID NOs:22, 23 and 24, respectively.
 4. The method of claim 1, wherein the Her-3 antibody is at least one antibody comprising an amino acid sequence set forth in SEQ ID NOs:9 and 11 for the light and heavy chains, respectively, or SEQ ID NOs:10 and 12 as set forth in Table 1 for the light and heavy chains, respectively.
 5. The method of claim 1, wherein the method comprises mixing the sample with the Her-3 antibody and determining the presence and/or quantity of the Her-3 antibody bound to Her-3 and/or Her-3 in a complex.
 6. The method of claim 1, wherein the method comprises measuring and/or quantifying the presence and/or amount of at least one of total Her-3, Her-3 homodimers, or Her-3 heterodimers.
 7. The method of claim 1, wherein the method comprises the steps of: (a) contacting the sample with (i) a proximity probe having an effective proximity and being capable of binding Her-3 and (ii) a binding compound having one or more signaling molecules attached thereto and being capable of binding Her-3, wherein binding of the proximity probe and the binding compound within the effective proximity produces a signal from the molecular tags that correlates with the presence and/or quantity of Her-3 and/or Her-3 in a complex; and (b) detecting the signal from the molecular tags to determine the presence and/or quantity of Her-3 and/or Her-3 in a complex.
 8. The method of claim 7, wherein the proximity probe and/or the binding compound comprise a Her-3 antibody.
 9. The method of claim 7, wherein the proximity probe or the binding compound comprises the Her-3 antibody that specifically binds to a polypeptide sequence set forth in one of SEQ ID NOs:1-8.
 10. The method of claim 7, wherein the proximity probe or the binding compound comprises a Her-3 antibody that specifically binds to a polypeptide sequence set forth SEQ ID NO:6.
 11. The method of claim 1, wherein the method comprises the steps of: (a) contacting the sample with a tagged Her-3 binding composition that specifically binds to Her-3 and has one or more molecular tags attached thereto via a cleavable linkage; (b) contacting the sample with a cleaving agent; (c) cleaving the cleavable linkage of the tagged Her-3 binding composition, thereby releasing the one or more molecular tag; and (d) quantitating the released molecular tags to determine the presence and/or amount of Her-3 in the sample.
 12. The method of claim 11, wherein the tagged Her-3 binding composition comprises a Her-3 antibody.
 13. The method of claim 11, wherein the tagged Her-3 binding composition comprises the Her-3 antibody that specifically binds to a polypeptide sequence set forth in one of SEQ ID NOs:1-8.
 14. The method of claim 1, wherein the measurement may be quantitative across a wide dynamic range.
 15. The method of claim 1, wherein the method comprises measuring and/or quantifying the presence and/or amount of Her-3 by immunohistochemistry.
 16. The method of claim 1, wherein the sample is an FFPE or solubilized FFPE sample.
 17. The method of claim 1, wherein the sample is a tumor sample.
 18. The method of claim 1, wherein the sample comprises a breast cancer sample, a colorectal cancer sample, an ovarian cancer sample, a non-small cell lung cancer sample, or a gastric cancer sample.
 19. The method of claim 1, wherein the sample comprises a breast cancer sample.
 20. The method of claim 1, wherein the sample comprises an early stage breast cancer sample or a metastatic breast cancer sample.
 21. The method of claim 1, wherein the sample comprises a Her-2 positive breast cancer sample.
 22. The method of claim 1, wherein subject is more likely to respond to a Her family targeted therapy, more likely to have a long time course during treatment with a Her family targeted therapy, and/or less likely to have a significant event during treatment with a Her family targeted therapy if the amount of Her-3 in the biological sample is below a Her-3 cutoff as compared to if the Her-3 in the biological sample is above the Her-3 cutoff.
 23. The method of claim 22, wherein the Her family targeted therapy comprises a multi-targeted agent or a single-targeted agent.
 24. The method of claim 22, wherein the Her family targeted therapy comprises a dual kinase inhibitor or a bispecific antibody.
 25. The method of claim 22, wherein the Her family targeted therapy comprises at least one of trastuzumab, lapatinib, pertuzumab, cetuximab, panitumumab, erlotinib, or gefitinib.
 26. The method of claim 22, wherein likeliness to respond, likeliness to have a long time course and/or likeliness to have a significant event is measured as at least one of overall survival rate, as time to progression, disease-free survival, progression-free survival, time to distant reoccurrence, hazard ratio and/or objective tumor response or clinical benefit using the RECIST criteria.
 27. An isolated antibody that specifically binds to a polypeptide sequence set forth in one of SEQ ID NOs:1-8.
 28. The antibody of claim 27, wherein the antibody is at least one of a monoclonal antibody comprising (a) a light chain variable region comprising CDR1, CDR2 and CDR3 having sequences as set forth in SEQ ID NOs:13, 14 and 15, respectively, and (b) a heavy chain variable region comprising CDR1, CDR2 and CDR3 having sequences as set forth in SEQ ID NOs:16, 17 and 18, or a monoclonal antibody comprising (a) a light chain variable region comprising CDR1, CDR2 and CDR3 having sequences as set forth in SEQ ID NOs:19, 20 and 21, respectively, and (b) a heavy chain variable region comprising CDR1, CDR2 and CDR3 having sequences as set forth in SEQ ID NOs:22, 23 and 24, respectively.
 29. The antibody of claim 27, wherein the antibody is an antibody comprising the amino acid sequence set forth in SEQ ID NOs:9 and 11 for the light and heavy chains, respectively, or an antibody comprising the amino acid sequence set forth in SEQ ID NOs:10 and 12 for the light and heavy chains, respectively.
 30. The method of claim 27, wherein the antibody specifically binds to a polypeptide sequence set forth in SEQ ID NO:6. 